Methods and compositions for detecting and modulating o-glycosylation

ABSTRACT

The invention relates to methods and products for modulating glycosylation of proteins. The invention is useful for identifying therapeutic compounds to treat glycosylation-associated disorders such as neurodegeneration, diabetes, including complications of diabetes such as insulin resistance, nephropathy, microvascular damage, and endothelial dysfunction. The invention is also useful for identifying therapeutic compounds to treat de-glycosylation-associated disorders such as ischemic damage and traumatic injury. The invention also relates in part to assays that are useful for identifying and testing candidate compounds for modulating glycosylation of proteins and also relates in part to compounds to treat glycosylation-associated diseases and disorders.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S. §119 (e) of U.S.Provisional Patent Application Ser. No. 60/934,803 filed Jun. 15, 2007,the entire contents of which is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under grant numberA144854 awarded by the National Institutes of Health. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The invention relates, in part, to methods and products for assessingO-glycosylation of polypeptides and for identifying compounds thatmodify O-glycosylation of polypeptides. The invention also relates, inpart, to O-glycosylation inhibitor compounds that have been identifiedand the use of such compounds for treatment ofO-glycosylation-associated disorders such as neurodegeneration, insulinresistance, diabetes, and complications of diabetes such as nephropathy,microvascular damage, and endothelial dysfunction. The invention alsorelates in part to assays and substrate polypeptides that are useful foridentifying and testing candidate compounds to determine their effect onmodulating glycosylation of polypeptides.

BACKGROUND OF THE INVENTION

The hexosamine biosynthetic pathway (HSP) is a minor branch of theglycolytic pathway, diverting 3-5% of cellular glucose toward thesynthesis of UDP-GlcNAc, which is either transported to the golgi andused in the synthesis of complex glycans or remains in the cytoplasmwhere it is the obligatory substrate for O-GlcNAc Transferase (OGT). OGTis the sole known enzyme to catalyze the transient glycosylation ofserine and threonine residues on many nuclear and cytoplasmic proteins(termed O-GlcNAcylation). This post-translational modification isdynamic and is a general method, like protein phosphorylation, of signaltransduction. There are also other glycosylating enzymes thatpost-translationally modify molecules in the body.

Excess flux through the HSP has been implicated in both early (insulinresistance) and late (nephropathy, microvascular damage) stages in thecourse of diabetes mellitus, both in vivo and in vitro. Diabetesinvolves a deficiency in the availability and/or utilization of insulin.Insulin is a hormone produced by the pancreas and is necessary for cellsto utilize glucose. Insulin resistance is a condition in which muscle,fat, and liver cells do not use insulin properly. As a result, thepancreas produces more insulin, which also cannot be properly used.Eventually, the pancreas cannot keep up with the body's need forinsulin, and excess glucose builds up in the bloodstream. Thus, ininsulin resistance, there may be high levels of blood glucose and highlevels of insulin circulating in the bloodstream at the same time.

Experiments have shown that insulin resistance due to increasedhexosamine flux is caused by hyper O-GlcNAcylation. Diabetics haveincreased production of two adipokines directly responsible for vascularinjury, plasminogen activator inhibitor-1 (PAI-1) and transforminggrowth factor β₁ (TGF-β1). Transcription of both of these proteins isdecreased in cell culture when levels of O-GlcNAcylation were decreased.The molecular mechanism for this is known; increased transcription ismediated by the O-GlcNAcylation state of the transcription factor Sp1.OGT activity and levels of O-GlcNAcylation and the activity ofadditional glycosylating enzymes have also been implicated in otherdisease states, such as Alzheimer's disease and cancer.

The ppGalNAcT family of glycosyltransferases initiates core-typeO-glycan formation, that may be elaborated further by glycan linkages,contributing to selectin ligand-dependent control of leukocytetrafficking, regulation of CD8+ T-cell aoptosis, and sensitivity tocolitis and preventing the onset of Tn syndrome. The prototype ppGalNACTfamily member is ppGalNAcT-1, which is expressed at high levels in mosttissues and cell types. Loss of ppGalNAcT-1 activity in a mouse modelresulted in bleeding disorder and impaired IgG production. ppGalNAcT-1has been shown to support normal homeostatic physiology and inflammatoryresponse [(Term et al., Mol Cell Biol 27: 8783-8796 (2007)].

SUMMARY OF THE INVENTION

The invention relates in part, to novel kinetic assays that can be usedto detect glycosylation of polypeptides and can be used to screencandidate compounds that modulate (e.g., inhibit or enhance)glycosylation of polypeptides. The invention also includes moleculesthat include detectably labeled polypeptide substrates that are preparedsuch that the polypeptide substrate includes a) an O-glycosylation siteand b) a specific protease cleavage site positioned in the polypeptidesuch that the rate of cleavage by the specific protease at the specificprotease cleavage site is different when the polypeptide is glycosylatedat the O-glycosylation site than when the polypeptide is notglycosylated at the O-glycosylation site. In addition, the detectablelabel on the polypeptide is positioned in the polypeptide such that achange in the detectable label identifies cleavage of the polypeptide bythe specific protease at the specific protease cleavage site. Suchpolypeptide substrates can be used in assays of the invention thatpermit detection of O-glycosylation of polypeptides and permit detectionof modulation of O-glycosylation by candidate modulatory compounds.

The invention also relates, in part, to newly identified compounds thatinhibit O-glycosylation of polypeptides. A number of compounds have nowbeen identified that inhibit O-glycosylation. O-glycosylation is atransient glycosylation of serine and/or threonine residues on nuclearand cytoplasmic proteins that is catalyzed by O-glycosylating enzymes.The newly identified compounds and analogs, derivatives, and variantsthereof may be useful for the treatment (including active and/orprophylactic treatment) of diseases and disorders associated withabnormal O-glycosylation. The invention includes, in part, methods fortreating diseases and conditions resulting from abnormal O-glycosylationand compositions for treating such diseases and conditions.

The assay also relates, in some aspects, to assays that are useful toidentify compounds (e.g., small molecules, etc) that inhibitO-glycosylation activity.

According to one aspect of the invention, isolated molecules areprovided. The molecules include a cleavable detectably labeledpolypeptide substrate having an O-glycosylation site and a specificprotease cleavage site positioned in the polypeptide such that the rateof cleavage by the specific protease at the specific protease cleavagesite is different when the polypeptide is glycosylated at theO-glycosylation site than when the polypeptide is not glycosylated atthe O-glycosylation site, wherein the detectable label is positioned inthe polypeptide such that a change in the detectable label identifiescleavage of the polypeptide by the specific protease at the specificprotease cleavage site. In some embodiments, the polypeptideO-glycosylation site is a serine or a threonine residue. In certainembodiments, the serine or threonine is positioned within 1, 2, 3, or 4amino acids of the protease cleavage site. In some embodiments, proteasecleavage site is a single site and there is only one specific proteasecleavage site. In some embodiments, the specific protease cleavage siteis positioned on the N-terminal side of the O-glycosylation site. Incertain embodiments, the specific protease cleavage site is positionedon the C-terminal side of the O-glycosylation site. In some embodiments,the detectable label includes a fluorescent, enzyme, radioactive,metallic, biotin, chemiluminescent, or bioluminescent molecule. In someembodiments, the detectable label is a fluorescent moiety. In certainembodiments, the change in the detectable label comprises a change influorescence of the fluorescent moiety. In some embodiments, thedetectable label is a FRET donor and a FRET acceptor pair. In someembodiments, the FRET donor and acceptor pair is7-diethylaminocoumarin-3-carboxylic acid (DEAC) and fluoresceinisothiocyanate; [2-(1-sulfonyl-5-naphthyl)-aminoethylamide] EDANS and4,4-dimethylazobenzene-4′carbonyl (DABCYL); fluorescein isothiocyanateand tetramethylrhodamine (TMR); Tryptophan or tyrosine and dinitrophenylmoieties; or a fluorescein isothiocyanate and a fluoresceinisothiocyanate. In some embodiments, the fluorescent moiety is a Cy5.5emitter or FITC, Texas red, tetramethylrhodamine (TMR), AlexaFluor dyes,HiLyte Fluorophores, or [2-(1-sulfonyl-5-naphthyl)-aminoethylamide]EDANS. In certain embodiments, the polypeptide further comprises aquenching moiety. In some embodiments, the quenching moiety is QXL-520™,BHQ3, Iowa black, 4,4-dimethylazobenzene-4′carbonyl (DABCYL), BHQ1,BHQ10, QXL-570, QXL-620, dinitrophenyl (DNP) containing groups, orQSY-7, 9-21, or 35. In some embodiments, the polypeptide comprises theamino acid sequence set forth as STPVSSANMK (SEQ ID NO:1). In certainembodiments, the polypeptide comprises the amino acid sequence set forthas STPVSFANMK (SEQ ID NO:2). In some embodiments, the polypeptidecomprises the amino acid sequence set forth as STPVSRANMK (SEQ ID NO:3).In some embodiments, the polypeptide comprises the amino acid sequenceset forth as EYIPTVFDNK (SEQ ID NO:4). In some embodiments, thepolypeptide comprises the amino acid sequence set forth as EYRPTVFDNK(SEQ ID NO:5). In certain embodiments, the polypeptide comprises theamino acid sequence set forth as EYIPTVDDNK (SEQ ID NO:6). In someembodiments, the polypeptide comprises the amino acid sequence set forthas EPGPTEAPK (SEQ ID NO:25). In some embodiments, the polypeptidecomprises the amino acid sequence set forth as EDAVTPGPK (SEQ ID NO:26).In some embodiments, the protease cleavage site is a proteinase Kcleavage site, a trypsin cleavage site, a chymotrypsin cleavage site, athermolysin cleavage site, Staphylococcal peptidase I cleavage site,Proline-endopeptidase cleavage site, Pepsin cleavage site, Glutamylendopeptidase cleavage site, Factor Xa cleavage site, Granzyme B Lysylendopeptidase cleavage site, Asp-N Endopeptidase cleavage site, orenterokinase cleavage site.

According to another aspect of the invention, methods of detectingO-glycosylation of a polypeptide are provided. The methods include (a)contacting a glucosyltransferase enzyme and a UDP-sugar with a moleculethat includes a detectably labeled polypeptide having: (i) anO-glycosylation site and (ii) a specific protease cleavage sitepositioned in the polypeptide such that the rate of cleavage of thepolypeptide by the specific protease at the specific protease cleavagesite is different when the polypeptide is O-glycosylated at theglycosylation site than when the polypeptide is not O-glycosylated atthe glycosylation site, wherein the detectable label is positioned inthe polypeptide such that a change in the detectable label identifiescleavage of the polypeptide by the specific protease; (b) adding to thecontacted polypeptide the specific protease that cleaves at the specificprotease cleavage site of the polypeptide; and (c) monitoring thecleavage of the polypeptide, wherein the rate of cleavage ischaracteristic of the level of O-glycosylation of the glycosylationsite. In some embodiments, the O-glycosylation site comprises a serineor threonine amino acid residue. In certain embodiments, monitoringcleavage comprises monitoring cleavage rate. In some embodiments,wherein the sugar is a UDP-GlcNAc, UDP-GalNAc, UDP-Gal, UDP-Glc,UDP-GlcA (UDP-glucuronic acid), GDP-fucose, CMP-sialic acid, orUDP-xylose. In some embodiments, the glucosyltransferase enzyme isO-GlcNAc transferase; an N-acetylgalactosaminyltransferase; UDP-D-xyloseproteoglycan core protein (3 D-xylosyltransferase, UDPGalNAc:polypeptide N-acetylgalactosaminyltransfersase, ppGalNAcTs,GDP-fucose protein O-fucosyltransferase 1 (POFUT1), UDP-glucose:proteinglucosyltransferase (glycogen initiator synthase orEGF-glucosyltransferase), or a Rho-glucosylating toxin (C. difficileToxin A and Toxin B).

According to yet another aspect of the invention, methods of identifyingan agent that modulates polypeptide O-glycosylation are provided. Themethods include (a) contacting a glucosyltransferase enzyme, a sugardonor, and a candidate modulating compound with a molecule that includesa detectably labeled polypeptide having: (i) a glycosylation site and(ii) a specific protease cleavage site positioned in the polypeptidesuch that the rate of specific protease cleavage of the polypeptideglycosylated at the glycosylation site is different than the rate ofspecific protease cleavage of the polypeptide not glycosylated at theglycosylation site, wherein the detectable label is positioned in thepolypeptide such that a change in the detectable label identifiescleavage of the polypeptide by the specific protease at the specificprotease cleavage site; (b) adding to the contacted polypeptide thespecific protease; (c) monitoring specific protease cleavage of thepolypeptide; and (d) comparing the specific protease cleavage of thepolypeptide to a control specific protease cleavage, wherein adifference in the control specific protease cleavage compared to thecleavage of the polypeptide contacted with the candidate modulatingcompound identifies the candidate modulating compound as modulatingO-glycosylation of the polypeptide. In certain embodiments theO-glycosylation site comprises a serine or threonine amino acid residue.In some embodiments the glucosyltransferase enzyme is O-GlcNActransferase; an N-acetylgalactosaminyltransferase; UDP-D-xyloseproteoglycan core protein β D-xylosyltransferase, UDP GalNAc:polypeptideN-acetylgalactosaminyltransfersase, ppGalNAcTs, GDP-fucose proteinO-fucosyltransferase 1 (POFUT1), UDP-glucose:protein glucosyltransferase(glycogen initiator synthase or EGF-glucosyltransferase), or aRho-glucosylating toxin (C. difficile Toxin A and Toxin B). In someembodiments, the sugar donors is UDP-GlcNAc, UDP-GalNAc, UDP-Gal,UDP-Glc, UDP-GlcA (UDP-glucuronic acid), GDP-fucose, CMP-sialic acid, orUDP-xylose. In some embodiments the sugar donor is a UDP-sugar. Incertain embodiments the control level of cleavage is the level ofspecific protease cleavage of a essentially equivalent polypeptide of(a) contacted with glucosyltranferase enzyme, the sugar donor, and thespecific protease; but not contacted with the candidate modulatingagent. In some embodiments monitoring cleavage comprises monitoring therate of cleavage. In some embodiments the specific protease cleavage ofthe polypeptide contacted with the candidate modulating agent isincreased compared to the control cleavage identifying the candidatemodulating agent as an inhibitor of the O-glycosylation of theO-glycosylation site.

According to another aspect of the invention, kits for identifying anagent that modulates O-glycosylation are provided. The kits include apackage housing a first container containing a polypeptide substrate ofany forgoing aspects of the invention, and instructions for using thepolypeptide to identify modulators of O-glycosylation. In certainembodiments, the kit also includes a second container containing thespecific protease that cleaves at the specific protease site of thepolypeptide. In some embodiments, the polypeptide comprises the aminoacid sequence set forth as one of SEQ ID NOs:1-6, 25, or 26.

According to yet another aspect of the invention, compositions thatinclude an isolated compound of the compounds set forth as compounds1-28 or a derivative, analog, or variant of one of compounds 1-28 and apharmaceutically acceptable carrier are provided. The invention alsoprovides compositions that include other isolated compounds that aredisclosed herein and act as inhibitors of O-linked glycosylation. Insome embodiments, an inhibitor compound of the invention may be providedin a composition that also includes a pharmaceutically acceptablecarrier.

According to another aspect of the invention, methods for treating anO-glycosylation-associated disease or condition in a subject areprovided. The methods include administering to a subject in need of suchtreatment an effective amount of an O-glycosylation inhibiting compoundto treat the O-glycosylation-associated disease or condition, whereinthe O-glycosylation-inhibiting compound is any one of compounds 1-28 oran analog, derivative, or variant of any one of compounds 1-28 thatinhibits O-glycosylation activity. In some embodiments, the subject ishuman. In certain embodiments, the O-glycosylation-inhibiting compoundis linked to a targeting molecule. In some embodiments, theO-glycosylation-inhibiting compound is administered prophylactically toa subject at risk of having an O-glycosylation-associated disease ordisorder. In some embodiments, the O-glycosylation-inhibiting compoundis administered in combination with an additional drug for treating anO-glycosylation-associated disease or disorder. In some embodiments, theO-glycosylation-associated disease or disorder is Alzheimer's disease;cancer; diabetes mellitus, insulin resistance, a complication ofdiabetes, tumorigenesis, metastasis, bacterial infection and associatedcomplications such as sepsis. In certain embodiments, the complicationof diabetes is microvascular damage, insulin resistance, vasculardamage, nephropathy, skin ulcers, circulatory damage, diabeticnephropathy, diabetic retinopathy, macro-vascular disease,micro-vascular disease, or diabetic neuropathy. The invention alsoprovides methods of treating an O-glycosylation-associated disease orcondition that includes administration of one or more compoundsdisclosed herein that act as inhibitors of O-linked glycosylation.

According to another aspect of the invention, kits for treating asubject in accordance with any embodiments of the forgoing aspect of theinvention are provided. The kits include (a) a package housing a firstcontainer containing at least one dose of an O-glycosylation-inhibitingcompound, and (b) instructions for using the O-glycosylation-inhibitingcompound in the treatment of an O-glycosylation-associated disease ordisorder. In some embodiments, the O-glycosylation-inhibiting compoundis one of the compounds set forth as compounds 1-28 or an analog,derivative, or variant of one of the compounds set forth as compounds1-28 that inhibits O-glycosylation activity. In some embodiments, theO-glycosylation-inhibiting compound is linked to a targeting molecule.In certain embodiments, the O-glycosylation-inhibiting compound isadministered prophylactically to a subject at risk of having anO-glycosylation-associated disease or disorder. In some embodiments, theO-glycosylation-inhibiting compound is administered in combination withan additional drug for treating an O-glycosylation-associated disease ordisorder. In some embodiments, the O-glycosylation-associated disease ordisorder is Alzheimer's disease; cancer; diabetes mellitus, insulinresistance, a complication of diabetes, tumorigenesis, metastasis,bacterial infection and associated complications such as sepsis. Incertain embodiments, the complication of diabetes is microvasculardamage, insulin resistance, vascular damage, nephropathy, skin ulcers,circulatory damage, diabetic nephropathy, diabetic retinopathy,macro-vascular disease, micro-vascular disease, or diabetic neuropathy.

According to yet another aspect of the invention, methods for inhibitingO-glycosylation activity in a cell or tissue are provided. The methodsinclude contacting the cell or tissue with an effective amount of anO-glycosylation-inhibiting compound to inhibit O-glycosylation activityin the cell, or tissue. In some embodiments, theO-glycosylation-inhibiting compound is one of the compounds set forth ascompounds 1-28 or an analog, derivative, or variant of one of thecompounds set forth as compounds 1-28 that inhibits O-glycosylationactivity. In some embodiments, the O-glycosylation-inhibiting compoundis linked to a targeting molecule.

According to yet another aspect of the invention, methods of detectingde-glycosylation of a polypeptide are provided. The methods include (a)contacting a glycosidase enzyme with a molecule that includes adetectably labeled polypeptide substrate having: (i) a glycosylatedO-glycosylation site and (ii) a specific protease cleavage sitepositioned in the polypeptide such that the rate of cleavage of thepolypeptide by the specific protease at the specific protease cleavagesite is different when the polypeptide is O-glycosylated at theglycosylation site than when the polypeptide is not O-glycosylated atthe glycosylation site, wherein the detectable label is positioned inthe polypeptide such that a change in the detectable label identifiescleavage of the polypeptide by the specific protease; (b) adding to thecontacted polypeptide the specific protease that cleaves at the specificprotease cleavage site of the polypeptide; and (c) monitoring thecleavage of the polypeptide, wherein the rate of cleavage ischaracteristic of the level of de-glycosylation of the glycosylationsite. In certain embodiments, the O-glycosylation site includes a serineor threonine amino acid residue. In some embodiments, monitoringcleavage comprises monitoring cleavage rate. In some embodiments, theglycosidase enzyme is N-acetyl β-glucosaminidase.

According to another aspect of the invention, methods for identifying anagent that modulates polypeptide de-glycosylation are provided. Themethods include (a) contacting a glycosidase enzyme and a candidatemodulating compound with a molecule that includes a detectably labeledpolypeptide substrate having: (i) an O-glycosylated glycosylation siteand (ii) a specific protease cleavage site positioned in the polypeptidesuch that the rate of specific protease cleavage of the polypeptideglycosylated at the glycosylation site is different than the rate ofspecific protease cleavage of the polypeptide not glycosylated at theglycosylation site, wherein the detectable label is positioned in thepolypeptide such that a change in the detectable label identifiescleavage of the polypeptide by the specific protease at the specificprotease cleavage site; (b) adding to the contacted polypeptide thespecific protease; (c) monitoring specific protease cleavage of thepolypeptide; and (d) comparing the specific protease cleavage of thepolypeptide to a control specific protease cleavage, wherein adifference in the control specific protease cleavage compared to thecleavage of the polypeptide contacted with the candidate modulatingcompound identifies the candidate modulating compound as modulatingde-glycosylation of the polypeptide. In some embodiments, theO-glycosylation site comprises a serine or threonine amino acid residue.In certain embodiments, the glycosidase enzyme is N-acetylβ-glucosaminidase. In some embodiments, the control level of cleavage isthe level of specific protease cleavage of a essentially equivalentpolypeptide of (a) contacted with the glycosidase enzyme and thespecific protease; but not contacted with the candidate modulatingagent. In some embodiments, monitoring cleavage comprises monitoring therate of cleavage. In certain embodiments, the specific protease cleavageof the polypeptide contacted with the candidate modulating agent isdecreased compared to the control cleavage identifying the candidatemodulating agent as an inhibitor of the de-glycosylation of theO-glycosylation site.

According to yet another aspect of the invention, kits identifying anagent that modulates de-glycosylation are provided. The kits include apackage housing a first container containing a polypeptide substrate ofany embodiment of a foregoing aspect of the invention, and instructionsfor using the polypeptide to identify modulators of de-glycosylation. Insome embodiments, additionally including a second container containingthe specific protease that cleaves at the specific protease site of thepolypeptide. In some embodiments, the polypeptide comprises the aminoacid sequence set forth as one of SEQ ID NOs:1-6, 25, or 26.

These and other objects of the invention will be described in furtherdetail in connection with the detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of a FRET and a quencher/fluorescencepair detectable labels.

FIG. 2 shows structures of polypeptides 4.1, 4.4, and 4.5 and theirrelative glycosylation rate with polypeptide 4.1 having as the natural(wild-type) sequence having a glycosylation rate of 100%. (Polypeptide4.1 S→S (SEQ ID NO:1); Polypeptide 4.2 S→F (SEQ ID NO:2); Polypeptide4.5 S→R (SEQ ID NO:3)).

FIG. 3 shows graphs demonstrating proteolysis of glycosylated (squares)and un-glycosylated (diamonds) polypeptide 4.8 (SEQ ID NO: 3).Proteolysis with serial dilutions of trypsin (FIG. 3B) and proteinase K(FIG. 3A) are shown, with the cleavage site of each peptide shown above(FITC-AhaSTPV (SEQ ID NO:21); SRANMK-DEAC (SEQ ID NO:22); FITC-AhaSTPVSR(SEQ ID NO:23); ANMK-DEAC (SEQ ID NO: 24). The x-axis of both graphs ismg/mL protease concentration and the y-axis is the 535/460 emissionratio.

FIG. 4 shows a scatter plot of 535/460 emission ratio of polypeptide 4.5in the presence of known bioactive small molecules. The drop in ratiofor ˜5% of these compounds is dues to compound autofluorescence.

FIG. 5 shows three large classes of autofluorescent compounds. Thenumber and percentage below each compound is the number of members ofeach class represented in the ICCB library and percent of the totallibrary. X═C,O,N.

FIG. 6 shows a scatter plot of 535 emission (RFU) of polypeptide 4.9 inthe presence of known bioactive small molecules. In this case,autofluorescence would lead to a reproducible increase in signal.

FIG. 7 is a graph showing 535 nm emission (RFU) of mixtures ofglycosylated and unglycosylated polypeptide 4.9. RFU is shown on they-axis, and percent glycopeptide is shown on the x-axis. The slope andR² values are shown.

FIG. 8 shows a graph demonstrating the correlation between hit potenciesin the primary and secondary assay. Although some strong hits inhibitweakly in the secondary assay, in general the stronger the primary hit,the more potent the inhibitor.

FIG. 9 shows representative fluorescent compounds (identified here ascompounds i, ii, iii, and iv) found in the primary hit set.

FIG. 10 shows compounds, identified here as compounds 9, 3, 4, and 5,which were hits with reactive or potentially reactive functional groups.Schiff base hydrolysis unmasks an aldehyde (far right, compound 5).

FIG. 11 shows benzothiazole-6-carboxylic scaffold (at top) and compounds22, 23 and 1.

FIG. 12 shows 3-amino-4-phenylbutanoic acid scaffold (at top) andcompounds 19 and 12.

FIG. 13 shows compounds that were singleton hits with the assay. Toprow, left to right, shows→compounds 10, 2 and 16. Middle row, left toright shows compounds 17, 6, and 18. Bottom row, left to right, showscompounds 15 and 21.

FIG. 14 shows the biochemical activity of ppGalNAcTs

FIG. 15 shows the general substrate for ppGalNAcT (EPGPTEAPK SEQ ID NO:25) in FIG. 15A and in FIG. 15B shows the selective substrate for ppGalNAcT1 (EDAVTPGPK SEQ ID NO:26).

FIG. 16 shows the protease protection assay for ppGalNacT1: Substratepeptide EPGPTEAPK (SEQ ID NO: 25); Cleaved substrate peptides EPGPT (SEQID NO: 27) and EAPK (SEQ ID NO: 28).

FIG. 17 shows the results for the protease protection assay forppGalNacT1. FIG. 17A shows the assay window and FIG. 17B shows the assaylinearity graph.

FIG. 18 shows in FIG. 18A the % of glycosylation in the presence ofcompound 24 showing that compound 24 is a general ppGalNAcT inhibitorand inhibits both the T2 and T1 ppGalNAcT. FIG. 18B shows the %glycosylation in the presence of compound 25, which is more selectivelyinhibits ppGalNAcT2 than ppGalNAcT1.

FIG. 19 shows selected ppGalNACT small molecule inhibitors (left toright: Compound 26, compound 24, and compound 27)

FIG. 20 shows the results of growing ppGALNAcT2 crystals in the absenceof an inhibitor (FIG. 20A) and in the presence of an inhibitor FIG. 20B.

DETAILED DESCRIPTION OF THE INVENTION

Methods and products of the invention, in part, involve assays todetermine O-glycosylation of polypeptides and to identify compounds thatmodulate activity of enzymes that O-glycosylate polypeptides. Theinvention also includes compounds, and compositions that containcompounds that modulate O-glycosylation of polypeptides. In someembodiments, the modulation is inhibition and the compounds may reduceO-glycosylation of polypeptides by an O-glycosylating enzyme. In certainembodiments, the inhibition may be enhancement of O-glycosylation andthe compound may increase the O-glycosylation of polypeptides by anO-glycosylating enzyme. Methods of the invention may includeadministration of one or more compounds that modulate O-glycosylation ofan O-glycosylation site in a polypeptide. For example, a compounds ofthe invention include compounds that modulate O-glycosylation of serineand/or threonine in a polypeptide.

O-glycosylating enzymes catalyze the transfer of the N-acetylglucosaminefrom an activated sugar donor to a serine or threonine residue in apolypeptide, e.g. to a protein or polypeptide substrate. Examples ofO-glycosylating enzymes whose activity may be monitored using assays ofthe invention, and whose activity may be modulated by compounds of theinvention, include, but are not limited to, the enzymes O-Glc-NActransferase (OGT), N-acetylgalactosaminyltransferaseUDP-D-xylose:proteoglycan core protein beta-D-xylosyltransferase; UDPGalNAc:polypeptide N-acetylgalactosaminyltransferases: ppGalNAcTs,GDP-fucose protein O-fucosyltransferase 1 (POFUT1), UDP-glucose:proteinglucosyltransferase (glycogen initiator synthase OREGF-glucosyltransferase); and Rho-glucosylating toxins such as C.difficile Toxin A and Toxin B, etc. The invention, in some aspects, alsoincludes the use of O-glycosylation enzyme splice variants. Non-limitingexamples of sugar donors useful in methods of the invention include:UDP-GlcNAc, UDP-GalNAc, UDP-Gal, UDP-Glc, UDP-GlcA (UDP-glucuronicacid), GDP-fucose, CMP-sialic acid, and UDP-xylose.

Compositions of the invention include compounds that modulateO-glycosylation activity in cells, tissues, and subjects. As usedherein, the term “O-glycosylation-inhibiting compound” means a compoundthat reduces O-glycosylation of serine and/or threonine residues in apolypeptide. Methods of the invention, in some aspects, involve theadministration of one or more O-glycosylation-inhibiting compounds to acell, tissue, or subject and are useful to reduce or prevent cell deathand/or damage or disease associated with hyper O-glycosylation ofpolypeptides such as that resulting in an O-glycosylation-associateddisease or disorder. In some embodiments, methods of the inventioninclude administration of one or more de-glycosylation inhibitingcompounds to a cell, tissue, or subject and are useful to reduce damageor disease that is associated with hypo O-glycosylation of polypeptidessuch as that resulting from an de-glycosylation-associated disease ordisorder. The terms “O-glycosylation” and “glycosylation” are usedinterchangeably herein and the terms “protein,” “polypeptide,” and“peptide” are used interchangeably.

Diseases and Disorders

As used herein, the term “O-glycosylation-associated disease ordisorder” includes, but is not limited to diseases and disorders inwhich there is abnormal O-glycosylation enzyme activity and/or abnormallevels of O-glycosylation. As used herein, the term “O-glycosylationenzyme activity” means O-glycosylation enzyme-mediated O-glycosylation.In some disease and conditions an abnormal level of O-glycosylationenzyme activity and/or O-glycosylation of a polypeptide may be a levelthat is higher than a normal level (hyper-O-glycosylation). In certaindiseases or conditions an abnormal level of O-glycosylation may be alevel that is lower than a normal level (hypo-O-glycosylation). As usedherein, a “normal” level of O-glycosylation is the level ofO-glycosylation in a cell, tissue, or subject that does not have adisease or disorder associated with O-glycosylation enzyme activity,glycosylase enzyme activity, and/or with the O-glycosylation state ofpolypeptides.

Examples of diseases and disorders associated with O-glycosylationenzyme activity and/or O-glycosylation of polypeptides include, but arenot limited to neurodegenerative disorders such as Alzheimer's disease;cancer; diabetes mellitus, insulin resistance, and complications ofdiabetes, tumorigenesis, metastasis, bacterial infection and associatedcomplications such as sepsis, and complications of otherO-glycosylation-associated diseases. As used herein, the term“complication of diabetes” is used to mean a disorder that is associatedwith diabetes. Non-limiting examples of complications of diabetesinclude microvascular damage, insulin resistance, vascular damage,nephropathy, skin ulcers, circulatory damage, diabetic nephropathy,diabetic retinopathy, macro-vascular disease, micro-vascular disease,and diabetic neuropathy. In some aspects of the invention, anO-glycosylation-inhibiting compound may be used to treat a subject withdiabetes.

The term “diabetic” as used herein, means a subject who, at the time thesample is taken, has a primary deficiency of insulin. The term diabeticincludes, but is not limited to, individuals with juvenile diabetes(Type 1 diabetes), adult-onset diabetes (Type 2 diabetes), gestationaldiabetes, and any other conditions of insulin deficiency and/orabnormally high levels of β-glycosylating enzyme activity and/orabnormally high levels of O-glycosylation of polypeptides. The terms“diabetic” and “diabetes” are terms of art, known and understood bythose practicing in the medical profession, a formal definition of whichcan be found in Harrison's Principles of Medicine (Harrisons, Vol 14,Principles of Internal Medicine, Eds. Fauci, A. S., E. Braunwald, K. J.Isselbacher, J. D. Wilson, J. B. Martin, D. L. Kasper, S. L. Hauser, D.L. Longo, McGraw-Hill, New York, 1999).

Subjects with blood glucose levels that are higher than normal but notyet in the range associated with a diagnosis of diabetes may beconsidered to have “pre-diabetes.” Pre-diabetes is also known in the artas “impaired fasting glucose” (IFG) or “impaired glucose tolerance”(IGT). Subjects with pre-diabetes have a higher risk of developing type2 diabetes, which is also known as adult-onset diabetes ornoninsulin-dependent diabetes. Subjects with pre-diabetes frequently goon to develop type 2 diabetes within 10 years, without intervention—suchas diet change and/or activity changes. Health effects associated withdiabetes may include heart attack, stroke, blindness, deafness,amputations, kidney failure, burning foot syndrome, venous insufficiencywith ulceration and stasis dermatitis. Subjects with pre-diabetes alsohave a higher risk of heart disease. Insulin resistance can also occurin people who have type 1 diabetes, especially if they are overweight.

Assays

In some aspects the invention includes assays to assess activity ofglycosidase enzymes and assays to identify compounds that modulate thedeglycosylation of polypeptides. De-glycosylation is the removal ofsugars from proteins. An example of a glycosidase enzyme, though notintended to be limiting is: N-acetyl β-glucosaminidase (EC 3.2.1.52).Assays of the invention may be used to identify compounds that modulatede-glycosylation of polypeptides. Compounds that modulatede-glycosylation of polypeptides are referred to herein asde-glycosylation modulating compounds. De-glycosylation modulatingcompounds of the invention may be de-glycosylation inhibitors (e.g.,glycosidase inhibitors) or de-glycosylation enhancers (e.g., glycosidaseenhancers). Such compounds may be administered to a cell, tissue, orsubject to prevent or treat de-glycosylation-associated diseases ordisorders. Diseases and disorders associated with de-glycosylation arereferred to herein as de-glycosylation-associated diseases and disordersand include, but are not limited to, ischemia and traumatic injury andcomplications thereof. De-glycosylation modulating compounds of theinvention may be used to treat or prevent ischemic and/or traumaticinjury. A de-glycosylation inhibitor may inhibit activity of aglycosylase enzyme.

Methods of the invention may include assays for determining activity ofO-glycosylating enzymes in the O-glycosylation of polypeptides. Assaysof the invention may also be used to assess whether or not a candidatecompound modulates O-glycosylation of polypeptides, e.g., whether itmodulates O-glycosylating enzyme activity.

Assays of the invention may include a molecule that comprises adetectably labeled polypeptide. The molecule may be a polypeptide or anyother type of molecule and includes a detectably labeled polypeptidesubstrate sequence. In some embodiments, the molecule consists only of adetectably labeled polypeptide substrate sequence. In other embodimentsof the invention the detectably labeled polypeptide may be part of alarger molecule that may have any structure or composition, as long asthe molecule can function and can be used in an assay of the invention.For example, a molecule used in an assay of the invention may be apolypeptide, the sequence of which includes but is longer than thedetectably labeled polypeptide substrate. Such a molecule may be used inmethods and assays of the invention as long as the molecule's structurepermits detection of O-glycosylation or de-glycosylation of the peptidesubstrate portion of the molecule in an assay of the invention.

Assays of the invention may include contacting a glycosylating enzymeand a sugar donor with a molecule comprising a polypeptide substrate ofthe invention under conditions that permit glycosylation of theglycosylation site in the polypeptide substrate. The polypeptidesubstrate is then contacted with a specific protease that recognizes thecleavage site for the specific protease that is included in thepolypeptide substrate. Because the rate of cleavage by the protease willdiffer depending on whether or not the polypeptide substrate isglycosylated at the glycosylation site, a determination can be madebased on the cleavage (e.g. rate of cleavage) whether the polypeptidesubstrate is or is not glycosylated. The rate of cleavage therefore isan indicator of glycosylation of the polypeptide substrate. This permitsthe assay to be used to determine whether a polypeptide substrate isglycosylated (e.g., whether a glycosylating enzyme has activity). Also,assays of the invention may be used to assess candidate compounds thatcan be included in the assay to see whether they modulate glycosylatingactivity and glycosylation. In some embodiments, an increase in cleavageby the protease in the presence of the candidate modulating compoundindicates that glycosylation is inhibited by the candidate modulatingcompound.

In some embodiments, assays of the invention may include contacting aglycosidase with a molecule that includes a polypeptide substrate of theinvention that is O-glycosylated at an O-glycosylation site in thepolypeptide substrate. The polypeptide substrate is then contacted witha specific protease that recognizes the cleavage site for the specificprotease that is included in the polypeptide substrate. Because the rateof cleavage by the protease will differ depending on whether or not thepolypeptide substrate is glycosylated at the glycosylation site, adetermination can be made based on the cleavage (e.g. rate of cleavage)whether the polypeptide substrate is or is not glycosylated. The rate ofcleavage therefore is an indicator of glycosylation of the polypeptidesubstrate. This permits the assay to be used to determine whether apolypeptide substrate contacted with a glycosidase has beende-glycosylated (e.g., whether the glycosidase was effective). Also,assays of the invention may be used to assess candidate compounds thatcan be included in the assay to see whether they modulate thede-glycosylating activity and de-glycosylation (e.g. whether theymodulate a glycosidase).

Polypeptide Substrates

A polypeptide substrate of the invention useful in methods and assays ofthe invention can be any suitable length for use in an assay, andincludes, at least, a) an O-glycosylation site and b) a specificprotease cleavage site. The O-glycosylation site consists of an aminoacid that can accept a sugar transferred by an O-glycosylating enzyme.Examples of O-glycosylation sites that may be included in a polypeptidesubstrate of the invention are the amino acids serine or threonine. Insome embodiments the polypeptide substrate is detectably labeled.

Methods and assays of the invention may be performed under conditionsthat permit glycosylation of the polypeptide substrate. For example,when an assay of the invention includes contacting a glucosyltransferaseenzyme and a sugar donor with a molecule that includes a detectablylabeled polypeptide substrate, the contact is carried out under suitableconditions for glycosylation of the polypeptide. Conditions, (e.g.,temperature, length of incubation, buffer constituents, pH, etc.) underwhich the contact is carried out are conditions under which theglucosylatranferase enzyme is able to utilize the sugar donor (e.g.,UDP-sugar or other suitable sugar donor) and glycosylate the polypeptidesubstrate at the O-glycosylation site. Such conditions are known in theart and may be optimized for a given glycosylating enzyme and sugarusing routine methods. Examples, though not intended to be limiting, ofconditions for glycosylating polypeptide substrates of the invention areprovided the Examples section herein. Alternative conditions that may beused with methods and compounds of the invention are known and may beselected by those of ordinary skill in the art. Methods and assays ofthe invention may also include the use of assays of the invention toassess the ability of candidate agents to modulate glycosylation of apolypeptide substrate of the invention. In such embodiments, an assaymay include conditions that are conducive to allowing glycosylation butmay also include a candidate compound to be tested for its ability tomodulate (inhibit or enhance) O-glycosylation.

Polypeptide substrates of the invention also include a specific proteasecleavage site that is positioned in the polypeptide substrate such thatthe rate of cleavage by the specific protease at the specific proteasecleavage site is different when the polypeptide is glycosylated at theO-glycosylation site than the rate of cleavage when the polypeptide isnot glycosylated at the O-glycosylation site. As used herein, the term“positioned” means placed in the amino acid sequence of the polypeptidesubstrate. In some embodiments, a specific protease cleavage site may belocated adjacent to the O-glycosylation site (e.g. directly next to theserine or threonine). In some embodiments, the specific proteasecleavage site may be located within 1, 2, 3, 4, or 5 amino acids of theO-glycosylation site. The specific protease cleavage site may be locatedon either the N-terminal side of the O-glycosylation site or on theC-terminal side of the O-glycosylation site. The protease cleavage sitemay be on the N-terminal side of the O-glycosylation site, which meansthe protease cleavage site is closer to the N terminus of thepolypeptide substrate than is the O-glycosylation site. Similarly, insome embodiments the protease cleavage site may be on the C-terminalside of the O-glycosylation site, which means the protease cleavage siteis closer to the C terminus of the polypeptide substrate than is theO-glycosylation site. Thus, in some embodiments of the invention theposition of the specific protease cleavage site may be exemplified asfollows:

X_(m)*X₀₋₄S/TX_(n) or X_(n)S/TX₀₋₄*X_(m),

where m and n are independently any number of amino acids 1 or higher, Xcan be any amino acid, with the number of amino acids determined by thesubscript, * is the specific protease cleavage site, and S/T is eitherserine or threonine. In some embodiments of the invention there is onlya single specific cleavage site in the polypeptide substrate. There maybe other of the specific cleavage sites outside of the polypeptidesubstrate as long as cleavage at the additional sites does not interferewith detection of cleavage of the polypeptide substrate. In certainembodiments, there is only one of the specific protease cleavage sitesin the polypeptide substrate. In some embodiments of the invention,there is only one of the specific protease cleavage sites between twodetectable labels or between a detectable label and its quenchingpartner in the polypeptide substrate.

As used herein the term “specific protease cleavage site” means thespecific location that the protease cleaves the polypeptide. Thus, thespecific protease cleavage site is located between two adjacent aminoacids. It will be understood by those of ordinary skill in the art thatthe recognition site for a specific protease may include one, two,three, four, or more amino acids depending on the specific recognitionrequirements of the protease for the polypeptide. Those of ordinaryskill in the art will readily be able to identify cleavage sites andrecognition sites for specific proteases. Details of specific proteasecleavage sites and protease recognition sites are well known in the art,including for example, at the PeptideCutter pages of ExPASY.(www.expasy.ch/tools/peptidecutter/) and Keil, B. Specificity ofproteolysis. Springer-Verlag Berlin-Heidelberg-NewYork, pp. 335. (1992).Those of ordinary skill in the art can use available tools and publishedinformation to identify a specific protease site for inclusion in apolypeptide substrate for use in methods and compositions of theinvention. Examples of proteases that may be used in methods of theinvention, include, but are not limited to: proteinase K, trypsin,chymotrypsin, thermolysin, Staphylococcal peptidase I,Proline-endopeptidase, Pepsin, Glutamyl endopeptidase, Factor Xa,Granzyme B Lysyl endopeptidase, Asp-N Endopeptidase, and enterokinase,etc. Those of skill in the art will be able to utilize additionalproteases cleavage sites in polypeptide substrates of the invention andwill be able to determine glycosylation and de-glycosylation status ofpolypeptides based on cleavage by specific proteases in methods of theinvention without undue experimentation.

Methods and assays of the invention may be performed under standardconditions that permit cleavage of the specific protease cleavage siteby the specific protease. For example, when an assay of the inventionincludes contacting a polypeptide substrate with a protease, the contactis carried out under suitable standard conditions that permit thespecific protease to function. Conditions, (e.g., temperature, length ofincubation, buffer constituents, pH, etc.) under which the contact iscarried out are conditions under which the protease is able to cleave apolypeptide substrate at the specific protease cleavage site. Suchstandard conditions are known in the art and may be optimized for thespecific protease using routine methods. Examples, though not intendedto be limiting, of conditions for protease cleavage are provided theExamples section herein. Alternative conditions that may be used withmethods and compounds of the invention are known and may be selected bythose of ordinary skill in the art without undue experimentation. Insome embodiments, glycosylation of a polypeptide substrate may reduce oreliminate cleavage by a specific protease under standard conditions thatpermit normal cleavage of the polypeptide substrate by the specificprotease.

In methods and molecules of the invention, the positioning of theO-glycosylation site in relation to the specific protease cleavage sitein the polypeptide substrate is such that the rate of cleavage of thepolypeptide substrate by the specific protease is different with thepolypeptide is glycosylated at the O-glycosylation site than the rate ofcleavage is with the O-glycosylation site is not glycosylated. Thus, thepresence of the O-glycosylated serine or threonine alters the rate ofcleavage of the polypeptide substrate by the specific protease. In someembodiments of the invention, the rate of cleavage of the polypeptidesubstrate is higher when the polypeptide is not glycosylated at theO-glycosylation site than when the polypeptide substrate is glycosylatedat the O-glycosylation site. Thus, in some embodiments, the differencein rate of cleavage indicates whether a polypeptide substrate (or aplurality of the polypeptide substrate) is glycosylated or is notglycosylated at the O-glycosylation site. For example, the rate ofcleavage by a specific protease at the specific protease cleavage sitemay be 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400,500, 600, or more times faster (including all times numbers in betweenthe listed numbers) in un-glycosylated versus glycosylated polypeptidesubstrates of the invention.

Detection

Detection of cleavage and cleavage rate of a polypeptide substrate ofthe invention can be performed using numerous methods. Polypeptidelength and the presence of fragments may be assessed as an indication ofcleavage. Such assessment may be done using polypeptide substrates thatare detectably labeled but methods of the invention may also be usedwith polypeptide substrates that are not detectably labeled, for exampleassessment of cleavage may be performed using assay methods such as gelelectrophoresis, centrifugation separation, etc. Those of ordinary skillin the art will recognize numerous methods available that can be used todetermine substrate length or the presence and/or identify of cleavagefragments.

In some embodiments of the invention, polypeptide substrates aredetectably labeled and detection of the detectable labels is used todetermine cleavage and/or rate of cleavage of a polypeptide substrate. Adetectable label on a polypeptide substrate may be positioned in thepolypeptide such that a change in the detectable label identifiescleavage of the polypeptide by the specific protease at the specificprotease cleavage site, and as indicated elsewhere herein, cleavage ofthe polypeptide substrate (e.g. rate of cleavage) indicates whether thepolypeptide substrate is glycosylated or not glycosylated at theO-glycosylation site.

The use of detectable labels for determining cleavage is well known bythose of ordinary skill in the art. A detectable moiety may bepositioned at one location on a polypeptide substrate such that removalof the label from the remainder of the substrate by cleavage of thesubstrate indicates cleavage of the substrate. A variety of methods maybe used to detect the label, depending on the nature of the label andother assay components. A detectable moiety may be directly detectedthrough optical or electron density, radioactive emissions, nonradiativeenergy transfers, etc. or indirectly detected with antibody conjugates,strepavidin-biotin conjugates, etc. Methods for detecting the labels arewell known in the art. In some embodiments, a detectable moiety may bebiotin, a fluorophore, chromophore, enzymatic, or a radioactive moiety.

In assays and methods of the invention, following a glycosylation stepand incubation with the protease specific for the protease site in thepolypeptide substrate, the rate of protease cleavage of the polypeptidemay be detected by any convenient method available to the user.Detection may be effected in any convenient way for the assay, forexample for a high-throughput assay detection may be most convenientusing a detectable label that is a fluorescent or luminescent moietythat allows for high sensitivity and rapid detection. In otherembodiments and assays, other types of detectable labels and detectionmethods may be used. A wide variety of detectable labels can be used,such as those that provide direct detection (e.g., radioactivity,luminescence, optical or electron density, etc.) or indirect detection(e.g., epitope tag such as the FLAG epitope, enzyme tag such ashorseradish peroxidase, etc.).

As used herein the term detectable label, includes, but is not limitedto a fluorescent or bioluminescent detectable moiety. Detection of afluorescent or bioluminescent detectable label of the invention may beperformed using any suitable imaging method, including, but not limitedto video microscopy, real-time imaging, or other means that permitimaging of detectable labels of the invention.

In some aspects of the invention, a detectable moiety is attached to apolypeptide substrate in a form that is not detectable, (e.g., isquenched or not fluorescent or luminescent at an appropriate detectionwavelength) until cleavage of the polypeptide substrate. For example, afluorescent moiety may be conjugated to one end of the polypeptidesubstrate and a light-quenching moiety such as BHQ3 or Iowa black may beconjugated on to another position of the polypeptide substrate, forexample with the protease cleavage site between the fluorescent andlight-quenching moieties. Thus, when the polypeptide substrate isintact, the fluorescence emitted from the fluorescent moiety is quenchedand when the polypeptide substrate is cleaved by the specific proteaseat the specific protease cleavage site, the fluorescence is unquenchedand may be detected. Similarly, FRET methods and molecules may be usedin methods and on polypeptide substrates of the invention such that adetectable shift in fluorescence occurs when the polypeptide substrateis cleaved at the specific protease cleavage site, thus separating thedonor and acceptor FRET molecules. The detectable label will bedetectable when the polypeptide substrate has been cleaved thusindicating cleavage at the protease cleavage site. FIG. 1 illustratesexamples of assays utilizing FRET moieties and quenching/fluorescentmoieties.

In some embodiments, a fluorescent or luminescent molecule is attachedto a polypeptide substrate (using standard methods) in conjunction withanother fluorescent molecule such that FRET and/or BRET methods resultin a wavelength of light emission that shifts when there is cleavage ofthe polypeptide substrate and the first fluorescent molecule is nolonger in close enough proximity to the second fluorescent molecule. Ineach case, a change in the level or wavelength of detectable lightemitted from the detectably labeled polypeptide substrate of theinvention upon cleavage by the specific substrate can be used to detectthe cleavage of the polypeptide substrate. As indicated above herein,the rate at which cleavage of a polypeptide substrate occurs indicatesthe glycosylation state of the O-glycosylation site of the polypeptidesubstrate. Thus, the rate of cleavage of a polypeptide substrate of theinvention indicates whether the O-glycosylation site on the polypeptidesubstrate is or is not glycosylated.

In some embodiments of the invention, detection of cleavage (and thusdetection of glycosylation or lack of glycosylation of a polypeptidesubstrate) is based on the appearance of a fluorescent or luminescentsignal that had originally been quenched, e.g., the unquenching of aquenched signal. As used herein, a quenching moiety is a quenchingmolecule that is attached to a polypeptide substrate of the invention.In some embodiments of the invention, a quenching moiety is anabsorbance moiety that does not fluoresce and is able to quench thefluorescent signal of the fluorescent moiety or detectable label. A darkquencher absorbs the fluorescent energy from the fluorophore, but doesnot fluoresce itself. Rather, the dark quencher dissipates the absorbedenergy, typically as heat. Non-limiting examples of dark ornon-fluorescent quenchers are Dabcyl, Black Hole Quenchers, Iowa Black,BH3Q, QSY-7, AbsoluteQuencher, Eclipse non-fluorescent quencher, andmetal clusters such as gold nanoparticles. Those of ordinary skill inthe art will be able to identify and use additional dark quenchers inthe methods of the invention without undue experimentation.

In some embodiments of the invention, detection of a target may be basedon a shift in fluorescence frequency of a fluorescent or luminescentmoiety of the cleaved polypeptide substrate. Examples of detectionmethods that utilize such a shift are FRET and BRET methods, both ofwhich are methods routinely used in the art. Thus, in some embodiments,a detectable label is a fluorescence donor or donor fluorophore and thequencher is a fluorescence acceptor or acceptor fluorophore. In someembodiments, the donor and acceptor fluorophores form a FRET(fluorescence resonance energy transfer) pair. If the donor fluorophoreis excited, for instance by a laser light, a portion of the energyabsorbed by the donor is transferred to acceptor fluorophore if theacceptor fluorophores are spatially close enough to the donor molecules(i.e., the distance between them must approximate or be less than theForster radius or the energy transfer radius). Once the acceptorfluorophore absorbs the energy, it in turn fluoresces in itscharacteristic emission wavelength, resulting in a shift in frequency offluorescence. Non-limiting examples of FRET donors include Alexa 488,Alexa 546, BODIPY 493, Oyster 556, Fluor (FAM), Cy3 and TMR (TAMRA).Examples of FRET acceptors include Cy5, Alexa 594, Alexa 647, Oyster656, Texas red, tetramethylrhodamine (TMR), AlexaFluor dyes, HiLyteFluorophores, and [2-(1-sulfonyl-5-naphthyl)-aminoethylamide] EDANS.

FRET generally requires only one excitation source (and thus wavelength)and only one detector. The detector may be set to either the emissionspectrum of the donor or acceptor fluorophore. The detector is set tothe donor fluorophore emission spectrum if FRET is detected by quenchingof donor fluorescence. Alternatively, the detector is set to theacceptor fluorophore emission spectrum if FRET is detected by acceptorfluorophore emission. In some embodiments, FRET emissions of both donorand acceptor fluorophores can be detected. In still other embodiments,the donor is excited with polarized light and polarization of bothemission spectra is detected.

In other embodiments, the resonance energy transfer signal is due toluminescence resonance energy transfer (LRET; Mathis, G. Clin. Chem. 41,1391-1397, 1995) and the donor moiety is a luminescent moiety. In someembodiments the luminescent moiety is a chemiluminescent moiety (CRET;Campbell, A. K., and Patel, A. Biochem. J. 216, 185-194, 1983). In someembodiments the luminescent moiety is bioluminescent moiety (BRET; Xu,Y., Piston D. W., Johnson, Proc. Natl. Acad. Sci., 96, 151-156, 1999).

When the resonance energy signal is due to chemiluminescence, the donormoiety can be a lanthanide like Europium or Terbium. Furthermore, wherethe resonance energy signal is due to chemiluminescence, the donormoiety can be a lanthanide chelate such as DTPA-cytosine, DTPA-cs124,BCPDA, BHHCT, Isocyanato-EDTA, Quantum Dye, or W1024 and the acceptormoiety can be Cy-3, ROX or Texas Red. In some embodiments, due to therange of effective resonance energy transfer of the lanthanide chelate,multiple acceptor moieties may be employed. The donor moiety can be alanthanide chelate and the acceptor moiety can be a phycobiliprotein. Incertain embodiments, the phycobiliprotein is Red Phycoerythrin (RPE),Blue Phycoerythrin (BPE), or Allophycocyanin (APC).

In BRET, the donor protein is a bio-luminescent protein and the acceptorprotein is a fluorescent protein. Donor luminescent protein used in BRETmay include, but are not limited to Renilla luciferase or fireflyluciferase. In some embodiments the fluorescent acceptor protein in BRETis a green, red, cyan or yellow fluorescent protein.

The positioning of the detectable labels on the polypeptide substrate issuch that a change in the detection of the detectable label indicatescleavage of the substrate. Examples of effective distances between aquenching moiety and a detectable label (or between two members of aFRET or BRET pair) on a polypeptide substrate of the invention areprovided herein in the Examples section and those of ordinary skill inthe art will recognize routine methods to determine and to optimize thedistance between a detectable label and a quencher (or two fluorescentor luminescent labels) for use in methods and compositions of theinvention. The use of quenching and fluorescence pairs is well known inthe art and those of ordinary skill in the art will be able to utilizeand optimize the use of such pairs in methods of the invention withoutundue experimentation.

In some embodiments, the signal of a quenched detectable label isquenched by at least 1%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100%, including all percentages in between eachpercentage listed. In some embodiments a FRET partner or fluorescentlabel is on the N-terminal side of the protease cleavage site in thepolypeptide substrate and the other member of the FRET pair or aquenching moiety, respectively is on the C-terminal side of the proteasecleavage site. In some embodiments a FRET partner or fluorescent labelis on the C-terminal side of the protease cleavage site and the otherFRET partner or the quenching moiety, respectively, is on the N-terminalside of the protease cleavage site of the polypeptide substrate.

Assay Controls

The rate of cleavage of a polypeptide substrate of the invention can becompared to a control rate of cleavage as a determination of theglycosylation or de-glycosylation of the polypeptide substrate. Forexample, when testing a candidate compound for activity as aglycosylation enzyme inhibitor or as a glycosidase inhibitor, a testsample that includes the enzyme or inhibitor can be run in an assay ofthe invention and the rate of cleavage of the polypeptide substrate canbe compared to a control rate of cleavage. A difference can indicatewhether or not the candidate compound inhibited the activity of theenzyme.

In methods and assays of the invention that are used to evaluate aneffect of a candidate compound on O-glycosylating enzyme activity, theresults in a test sample may be compared to the results in a controlsample that is essentially identical to the test sample, but lacks thecandidate compound. Differences in the levels of glycosylation of aglycosylation site in a test and control sample may be compared as ameasure of effect of the candidate compound on the O-glycosylation ofthe polypeptide. Those of ordinary skill in the art will recognize themanner of using control values and samples in conjunction with assaysand methods of the invention.

Similarly, in some embodiments of methods and assays of the invention, acontrol amount or level of O-glycosylating enzyme activity may be theamount in an control sample that is substantially identical to a testsample, but the control sample lacks one or more constituents that areincluded in the test sample. For example, a test sample for an assay todetermine whether a candidate compound modulates O-glycosylation of apolypeptide, may include: a candidate inhibitor compound and (i) asubstrate polypeptide that includes an O-glycosylation site and (ii) aspecific protease cleavage site positioned in the polypeptide such thatthe rate of cleavage by the specific protease at the specific proteasecleavage site is different when the polypeptide is glycosylated at theO-glycosylation site than when the polypeptide is not glycosylated atthe O-glycosylation site, wherein the detectable is label positioned inthe polypeptide such that a change in the detectable label identifiescleavage of the polypeptide by the specific protease at the specificprotease cleavage site. A control sample may include (i) a substratepolypeptide that includes an O-glycosylation site and (ii) a specificprotease cleavage site positioned in the polypeptide such that the rateof cleavage by the specific protease at the specific protease cleavagesite is different when the polypeptide is glycosylated at theO-glycosylation site than when the polypeptide is not glycosylated atthe O-glycosylation site, wherein the detectable is label positioned inthe polypeptide such that a change in the detectable label identifiescleavage of the polypeptide by the specific protease at the specificprotease cleavage site, but may not include the candidate inhibitorcompound. The specific protease may be added to both the control andtest samples under essentially equivalent conditions that permitcleavage of the unglycosylated polypeptide at the protease cleavage siteand the rate of cleavage of the polypeptide in each sample may bedetermined. The rate of cleavage in the sample and the control can becompared to each other to determine whether the candidate modulatingcompound altered the level of glycosylation of the polypeptide, thusresulting in a difference in the rate of cleavage between the controland the sample tested.

Screening for Compounds

The invention further provides efficient methods of identifyingpharmacological compounds or lead compounds and compounds that inhibit(or enhance) O-glycosylating enzyme activity and/or O-glycosylation ofpolypeptides. Generally, the screening methods involve assaying forcompounds that modulate (enhance or inhibit) the level ofO-glycosylating enzyme activity and/or O-glycosylation of polypeptides.As will be understood by those of ordinary skill in the art, thescreening methods may measure the level of O-glycosylating enzymeactivity directly, (e.g. binding and/or catalytic activity). Examples ofscreening methods are provided in the Examples section. In addition,screening methods may be utilized that measure a secondary effect ofO-glycosylating enzyme activity and/or O-glycosylation of polypeptides,for example, the level of cell damage and/or cell death in a cell ortissue sample or by measuring physiological and/or behavioralcharacteristics of an O-glycosylation-associated disease.

A wide variety of assays for O-glycosylation-inhibiting compounds can beused in accordance with this aspect of the invention, including,O-glycosylating enzyme activity assays, O-glycosylating enzymedisplacement assays, purified enzyme assays, cell-free assays,cell-based assays, cell-viability assays, etc. An example of such anassay that is useful to test candidate O-glycosylation-inhibitingcompounds is a purified enzyme assay provided in the Examples section.In assays for O-glycosylating enzyme activity modulating compounds, theassay mixture comprises a candidate compound. Typically, a plurality ofassay mixtures is run in parallel with different compound concentrationsto obtain a different response to the various concentrations. Typically,one of these concentrations serves as a negative control, i.e., at zeroconcentration of compound or at a concentration of agent below thelimits of assay detection.

It is contemplated that cell based assays can also be performed toassess the ability of compounds of the invention to inhibitO-glycosylation activity. Such cell-based assays can be performed usingcell samples and/or cultured cells. Biopsy cells and tissues as well ascell lines grown in culture are useful in the methods of the invention.

Candidate compounds useful in accordance with the invention encompassnumerous chemical classes, although typically they are organiccompounds. Preferably, the candidate compounds are small organiccompounds, i.e., those having a molecular weight of more than 50 yetless than about 2500, preferably less than about 1000 and, morepreferably, less than about 500. Candidate compounds comprise functionalchemical groups necessary for structural interactions with proteinsand/or nucleic acid molecules. The candidate compounds can comprisecyclic carbon or heterocyclic structure and/or aromatic or polyaromaticstructures substituted with one or more of the above-identifiedfunctional groups. Candidate compounds also can be biomolecules such aspeptides, saccharides, fatty acids, sterols, isoprenoids, purines,pyrimidines, derivatives or structural analogs of the above, orcombinations thereof and the like. Where the compound is a nucleic acidmolecule, the agent typically is a DNA or RNA molecule, althoughmodified nucleic acid molecules are also contemplated.

Candidate compounds are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides, synthetic organic combinatorial libraries,phage display libraries of random peptides, and the like. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are available or readily produced. Additionally,natural and synthetically produced libraries and compounds can bereadily be modified through conventional chemical, physical, andbiochemical means. Further, known pharmacological compounds may besubjected to directed or random chemical modifications such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs of the compounds. Candidate compounds also includeanalogs, derivatives, and/or variants of the O-glycosylation-modulatingcompounds described herein.

A variety of other reagents also can be included in the assay mixture.These include reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc. which may be used to facilitate optimalbinding, or to reduce non-specific or background interactions of thereaction components. Other reagents that improve the efficiency of theassay such as protease inhibitors, nuclease inhibitors, antimicrobialagents, and the like may also be used.

An exemplary purified enzyme assay that is a kineticO-glycosylation-inhibition assay is described in the Examples sectionherein. An O-glycosylation-inhibition assay may be used to assessO-glycosylation of a polypeptide and to identify candidate compoundsthat inhibit O-glycosylation of polypeptides and physiological effectsthereof.

In general, the mixture of the foregoing assay materials is incubatedunder conditions whereby, in the presence of the candidate compound, therate of cleavage of the polypeptide is enhanced or reduced, depending onwhether the candidate compound is a inhibitor or enhancing compound. Theorder of addition of components, incubation temperature, time ofincubation, and other parameters of the assay may be readily determinedby those of ordinary skill in the art. Such experimentation merelyinvolves optimization of the assay parameters, not the fundamentalcomposition of the assay. Incubation temperatures may vary at differentsteps of assays of the invention. For example, temperatures for theglycosylation step may differ from temperatures for the proteasecleavage step. Typically temperatures are between 4° C. and 40° C. andcan be optimized for the specific protease during protease cleavagesteps of the assay and can be independently optimized for the othersteps of the assay. Incubation times preferably are minimized tofacilitate rapid, high throughput screening, and typically are between0.1 and 10 hours.

Amino Acids and Sequences

The invention also includes in part, amino acid sequences and nucleicacid sequences that encode the amino acid sequences that are useful inthe assays of the invention. For example amino acid sequences ofpolypeptide substrates that are useful in assays of the invention. Asused herein the term ‘polypeptide substrate” means a polypeptide thathas: (a) an O-glycosylation site and (b) a specific protease cleavagesite positioned in the polypeptide such that the rate of cleavage by thespecific protease at the specific protease cleavage site is differentwhen the polypeptide is glycosylated at the O-glycosylation site thanwhen the polypeptide is not glycosylated at the O-glycosylation site,wherein the detectable is label positioned in the polypeptide such thata change in the detectable label identifies cleavage of the polypeptideby the specific protease at the specific protease cleavage site.Exemplary amino acid sequences of polypeptide substrates that are usefulin assays, methods, and compositions, of the invention include, but arenot limited to STPVSSANMK (SEQ ID NO:1) STPVSFANMK (SEQ ID NO:2);STPVSRANMK (SEQ ID NO:3); EYIPTVFDNK (SEQ IF NO:4); EYRPTVFDNK (SEQ IDNO:5); AND EYIPTVDDNK (SEQ ID NO:6); EPGPTEAPK (SEQ ID NO:25); EDAVTPGPK(SEQ ID NO:26). It will be understood that natural and non-natural aminoacids may be used in polypeptide substrate molecules of the invention.In some embodiments of the invention, all amino acids of a polypeptidesubstrate are naturally occurring amino acids. In some embodiments ofthe invention, a polypeptide substrate includes at least one natural andat least one non-natural amino acid. In certain embodiments, all aminoacids of a polypeptide substrate are non-naturally occurring aminoacids.

The invention, in some aspects, includes polypeptide substrates andnucleic acids that encode polypeptide substrates of O-glycosylation andalso includes homologs and alleles of the sequences. In general,homologs and alleles typically will share at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% nucleotide identity and/or atleast 95% amino acid identity to the sequences of an encoding nucleicacid and polypeptide substrate, respectively, in some instances willshare at least 95% nucleotide identity and/or at least 97% amino acididentity, in other instances will share at least 97% nucleotide identityand/or at least 98% amino acid identity, in other instances will shareat least 99% nucleotide identity and/or at least 99% amino acididentity, and in other instances will share at least 99.5% nucleotideidentity and/or at least 99.5% amino acid identity. The homology can becalculated using various, publicly available software tools developed byNCBI (Bethesda, Md.) that can be obtained through the internet.Exemplary tools include the BLAST system available from the website ofthe National Center for Biotechnology Information (NCBI) at the NationalInstitutes of Health. Pairwise and ClustalW alignments (BLOSUM30 matrixsetting) as well as Kyte-Doolittle hydropathic analysis can be obtainedusing the MacVector sequence analysis software (Oxford Molecular Group).Watson-Crick complements of the foregoing nucleic acids also areembraced by the invention.

The invention also includes degenerate nucleic acids that includealternative codons to those present in the native materials andmaterials of the invention. For example, serine residues are encoded bythe codons TCA, AGT, TCC, TCG, TCT and AGC. Each of the six codons isequivalent for the purposes of encoding a serine residue. Thus, it willbe apparent to one of ordinary skill in the art that any of theserine-encoding nucleotide triplets may be employed to direct theprotein synthesis apparatus, in vitro or in vivo, to incorporate aserine residue into an elongating polypeptide substrate. Similarly,nucleotide sequence triplets which encode other amino acid residuesinclude, but are not limited to: CCA, CCC, CCG, and CCT (prolinecodons); CGA, CGC, CGG, CGT, AGA, and AGG (arginine codons); ACA, ACC,ACG, and ACT (threonine codons); ARC and AAT (asparagine codons); andATA, ATC, and ATT (isoleucine codons). Other amino acid residues may beencoded similarly by multiple nucleotide sequences. Thus, the inventionembraces degenerate nucleic acids that differ from the biologicallyisolated nucleic acids in codon sequence due to the degeneracy of thegenetic code.

The invention also provides for polypeptide substrates that are modifiedfrom those specifically disclosed herein, and the use of suchpolypeptide substrates. Modified polypeptide substrate sequences mayinclude additions, substitutions, and/or deletions of one or more aminoacids. For use in methods and assays of the invention, the modifiedpolypeptides are detectably labeled and retain a glycosylation site andretain a protease specific site (which may differ from one exemplifiedin a polypeptide substrate disclosed herein), but the remaining aminoacids may be modified. The modified polypeptides retain the ability tobe glycosylated and to be cleaved by a protease in such a manner thatthe rate of cleavage of the polypeptide substrate indicates whether thepolypeptide substrate is or is not glycosylated. For example, modifiedpolypeptides having single amino acid changes can be prepared. Likewise,modified polypeptides having two amino acid changes can be prepared.Numerous modified polypeptides like these will be readily envisioned byone of skill in the art. Additional polypeptides having additionalsubstitutions (i.e., 3 or more), additions or deletions also can beprepared and are embraced by the invention as readily envisioned by oneof ordinary skill in the art. Any of the foregoing polypeptides can betested by routine experimentation for retention of the ability to beused as a polypeptide substrate in an assay of the invention. As usedherein the terms: “deletion”, “addition”, and “substitution” meandeletion, addition, and substitution changes to about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, or more amino acids of a polypeptide sequence of theinvention.

Compounds

Some assays of the invention can be used to monitor glycosylation ofpolypeptides and can also be used to assess whether or not a candidateagent can modulate activity of an O-glycosylation enzyme and todetermine whether an agent modulates O-glycosylation. Thus, assays andmethods of the invention may be used to identify compounds that areuseful for treating O-glycosylation-associated diseases or disorders.Deleterious effects seen in O-glycosylation-associated diseases and/ordisorders that are triggered by abnormal O-glycosylating enzyme activitymay be ameliorated by the administration of compounds and/orcompositions that modulate O-glycosylating enzyme activity. Compounds ofthe invention include compounds that modulate O-glycosylation enzymeactivity in the O-glycosylation of polypeptides in cells and/or tissues,thereby reducing the cell and tissue damage and clinical manifestationsof an O-glycosylation-associated disease or disorder. In someembodiments of the invention, the compounds inhibit activity of anO-glycosylating enzyme and reduce O-glycosylation. Table 1 providesexamples of O-glycosylation-inhibiting compounds of the invention.

TABLE 1 O-glycosylation-inhibiting compounds. Compounds 1-28

Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

Compound 7

Compound 8

Compound 9

Compound 10

Compound 11

Compound 12

Compound 13

Compound 14

Compound 15

Compound 16

Compound 17

Compound 18

Compound 19

Compound 20

Compound 21

Compound 22

Compound 23

Compound 24

Compound 25

Compound 26

Compound 27

Compound 28

Compounds presented herein as compounds 1-27 may be useful to inhibitO-glycosylation of proteins by O-glycosylating enzymes. Compounds suchas those exemplified by compounds 1-27 may be used to treat anO-glycosylation-associated disease or disorder in a subject in need ofsuch treatment. It will be understood by those of ordinary skill in theart that analogs, derivatives, or variants of one or more core compoundsor other compounds disclosed herein may be used as inhibitors ofO-glycosylation, and in some aspects of the invention, such inhibitorsmay be used to treat or prevent an O-glycosylation-associated disease orcondition, e.g., cancer, diabetes, pre-diabetes, etc.

Some assays of the invention can be used to monitor de-glycosylation ofpolypeptides and can also be used to assess whether or not a candidateagent can modulate activity of a glycosidase enzyme and to determinewhether an agent modulates de-glycosylation. Thus, assays and methods ofthe invention may be used to identify compounds that are useful fortreating de-glycosylation-associated diseases or disorders. Deleteriouseffects seen in de-glycosylation-associated diseases and/or disordersthat are triggered by abnormal de-glycosylating enzyme activity may beameliorated by the administration of compounds and/or compositions thatmodulate de-glycosylating enzyme activity. Compounds of the inventioninclude compounds that modulate glycosidase activity in thede-glycosylation of polypeptides in cells and/or tissues, therebyreducing the cell and tissue damage and clinical manifestations of ade-glycosylation-associated disease or disorder. In some embodiments ofthe invention, the compounds inhibit activity of a glycosidase andreduce de-glycosylation.

A compound of the invention may be an isolated compound. By “isolated”,it is meant present in sufficient quantity to permit its identificationor use according to the procedures described herein. Because an isolatedmaterial may be admixed with a carrier in a preparation or composition,such as, for example, for adding to a sample or for analysis, theisolated material may comprise only a small percentage by weight of thepreparation.

In some aspects of the invention, one or more of compounds 1-27 may beadministered to a subject that is free of indications for a previouslydetermined use of the compound(s). By “free of indications for apreviously determined use”, it is meant that the subject does not havesymptoms that call for treatment with one or more of the compounds ofthe invention for a previously determined use of that compound, otherthan the indication that exists as a result of this invention. As usedherein the term “previously determined use” of a compound means the useof the compound that was previously identified. Thus, the previouslydetermined use is not the use of inhibiting O-glycosylation enzymeactivity and/or the O-glycosylation of polypeptides.

Methods of the invention may include administration of anO-glycosylation-inhibiting compound that preferentially targets neuronalor vascular cells and/or tissues or other specific cell or tissue types.In addition, the compounds can be specifically targeted to neuronal orvascular tissue or other specific tissue types. Targeting may be doneusing various delivery methods, including, but not limited to:administration to neuronal or vascular tissue or other specific targettissue, and/or the addition of one or more targeting molecules to directthe compounds of the invention to neuronal or other tissues (e.g. glialcells, nerve cells, vascular cells, etc.). Additional methods tospecifically target compounds and compositions of the invention tospecific tissues, such as neuronal tissues, vascular tissues, or othertypes of tissues may also be used with the compounds and compositions ofthe invention, and are known to those of ordinary skill in the art.

The invention involves, in part, compounds that inhibit O-glycosylationactivity in cells, tissues, and/or subjects and use of such compounds toinhibit an O-glycosylation enzyme. The O-glycosylation inhibitors of theinvention may be used for treatment of cells, tissues, and/or subjectsand for research purposes. As used herein, the term “O-glycosylationactivity” means the O-glycosylation of polypeptides. It is understoodthat hyper O-glycosylation of polypeptides, which is the O-glycosylationof polypeptides at a level above a normal level, may occur in certaindiseases, including, but not limited to, diabetes and pre-diabetesconditions. The hyper O-glycosylation of polypeptides may also occur inother conditions and in other tissues as a result of disease and mayresult in cell and tissue damage. For example, levels of O-glycosylation(e.g., O-GlcNAcylation) that are above normal levels may result ininsulin resistance.

O-glycosylation-inhibiting compounds of the invention may beadministered to a subject to reduce the risk of anO-glycosylation-associated disorder. Reducing the risk of a disorderassociated with above-normal O-glycosylation means using treatmentsand/or medications to reduce O-glycosylating enzyme activity levels andor to reduce the level or O-glycosylation of polypeptides in thesubject, therein reducing, for example, the subject's risk ofAlzheimer's disease; cancer; diabetes mellitus, insulin resistance, acomplication of diabetes, tumorigenesis, metastasis, bacterial infectionand associated complications such as sepsis, vascular complicationsincluding but not limited to: diabetic nephropathy, diabeticretinopathy, macro-vascular disease, micro-vascular disease, anddiabetic neuropathy, etc.

As used herein, the term “subject” means any mammal. Subjects includebut are not limited to: humans, non-human primates, cats, dogs, sheep,pigs, horses, cows, rodents such as mice, rats, etc. In someembodiments, a subject is a mammal that may be in need of treatment withan O-glycosylation-modulating compound or a glycosylase-modulatingcompound of the invention. In some embodiments, assays of the inventionmay be run in vitro and compounds that inhibit O-glycosylation may betested and used in vitro in cells, for example in cultured cells.

As used herein the term “inhibit” with reference to O-glycosylationactivity means to reduce the amount of O-glycosylating enzyme activityand/or O-glycosylation of polypeptides a level or amount that isstatistically significantly less than an initial level, which may be acontrol level of O-glycosylation enzyme activity and/or O-glycosylation.As used herein, an initial level may be a level in a cell, tissue, orsubject not contacted with an O-glycosylation-inhibiting compound of theinvention. In some cases, the decrease in the level of O-glycosylationof polypeptides means the level of O-glycosylation is reduced from aninitial level to a level significantly lower than the initial level. Insome cases, the reduced level may be zero.

In some embodiments, a control level of O-glycosylating enzyme activityand/or O-glycosylation of polypeptides is the level that represents thenormal level of O-glycosylation enzyme activity and/or O-glycosylationin a cell, tissue, and/or subject. For example, a control level may be alevel that is not associated with hyper O-glycosylation and cell damageand/or death. In some instances, a control level will be the level in adisorder-free cell, tissue, or subject, that does not have abnormallyhigh levels of O-glycosylation enzyme activity (e.g. hyperO-glycosylation activity) and/or abnormally high levels ofO-glycosylation, and may be useful, for example, to monitor a change inthe level of O-glycosylating enzyme activity and/or O-glycosylation in acell. In other instances a control level of O-glycosylating enzymeactivity and/or O-glycosylation will be the level in a cell, tissue, orsubject with a disorder such as pre-diabetes or diabetes, etc. that isassociated with O-glycosylating enzyme activity and/or O-glycosylation,and may be useful, for example, to monitor a decrease in the level ofO-glycosylating enzyme activity and/or O-glycosylation of polypeptidesin a cell, tissue, or subject. In other embodiments, a control level ofO-glycosylating enzyme activity and/or O-glycosylation will be the levelin a cell, tissue, or subject with a disorder such as aneurodegenerative disorder, e.g. Alzheimer's disease, or cancer, etc.and may be useful, for example, to monitor a change in the level ofO-glycosylating enzyme activity and/or O-glycosylation in a cell,tissue, and/or subject. These and other types of control levels areuseful in assays to assess the efficacy of an O-glycosylatingenzyme-activity modulating and/or O-glycosylation modulating compound ofthe invention.

It will be understood by one of ordinary skill in the art that a controllevel of O-glycosylating enzyme activity and/or O-glycosylation may be apredetermined value, which can take a variety of forms. It can be asingle value, such as a median or mean. It can be established based uponcomparative groups, such as in disease-free groups that have normallevels of O-glycosylating enzyme activity and/or O-glycosylation ofpolypeptides. Other comparative groups may be groups of subjects withspecific disorders, e.g. pre-diabetes, insulin resistance, type 1diabetes, type 2 diabetes, complications of diabetes, neurodegenerativedisorders, Alzheimer's disease, cancer, etc. It will be understood thatdisease-free cells and/or tissues may be used as comparative groups forcells or tissues that have a O-glycosylating enzyme activity-relateddisorder and/or an O-glycosylation-associated disorder.

In some embodiments, a compound that inhibits and thereby reduces thelevel of O-glycosylating activity and/or O-glycosylation is a compoundthat reduces the likelihood or risk of having anO-glycosylation-associated disease or disorder. A level ofO-glycosylating enzyme activity and/or O-glycosylation in a cell,tissue, and/or subject may be one that is below the O-glycosylatingenzyme activity level in cells, tissues, and/or subjects with diabetesor pre-diabetes, e.g. may be a level that is clinically asymptomatic,but may still be treated and further reduced by administration of acompound of the invention. The invention relates in part to theadministration of an O-glycosylation-inhibiting compound of theinvention to a cell, tissue, and/or subject in an amount effective toreduce O-glycosylating enzyme activity and/or O-glycosylation ofpolypeptides in cells, tissues, and/or subjects with anO-glycosylation-associated disease or disorder.

Compound Analogs, Derivatives, Variants

In some aspects of the invention, O-glycosylation-modulating (inhibitingor enhancing) compounds include functional analogs, derivatives, and/orvariants of the O-glycosylation-modulating compounds of the inventionspecifically disclosed herein. Thus, the term“O-glycosylation-modulating compounds” may include functional analogs,derivatives, and/or variants of the compounds presented herein ascompounds 1-27. For example, functional analogs, derivatives, andvariants of the O-glycosylation-modulating compounds of Table 1 may bemade to enhance a property of a compound, such as stability. Functionalanalogs, derivatives, and variants of the compounds of Table 1 may alsobe made to provide a novel activity or property to a compound of Table1, for example, to enhance detection, to enhance potency, to reduce sideeffects, etc. In some embodiments of the invention, modifications to anO-glycosylation-modulating compound of the invention can be made to thestructure or side groups of the compound and can include one or moredeletions, substitutions, and additions of atoms, or side groups.Alternatively, modifications can be made by addition of a linkermolecule, addition of a detectable moiety, such as biotin or afluorophore, chromophore, enzymatic, and/or radioactive label, and thelike.

Analogs of the O-glycosylation-modulating compounds of Table 1 thatretain some or all of the O-glycosylation-modulating properties also canbe used in accordance with aspects of the invention. In someembodiments, an analog of a molecule may have a higher level ofO-glycosylation-modulating activity than the original compound. Chemicalgroups that can be added to or substituted in the molecules include:hydrido, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycyl, aryl,heteroaryl, alkoxy, aryloxy, sulfoxy, acyl, amino, acyloxy, acylamino,carboalkoxy, carboxyamido, carboxyamido, halo and thio groups.Substitutions can replace one or more chemical groups or atoms on themolecules provided herein, e.g., compounds 1-27. Examples of substitutedcompounds are provided in the Examples section.

Molecular terms, when used in this application, have their commonmeaning unless otherwise specified. The term “hydrido” denotes a singlehydrogen atom (H). The term “acyl” is defined as a carbonyl radicalattached to an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, arylor heteroaryl group, examples of such radicals being acetyl and benzoyl.The term “amino” denotes a nitrogen radical containing two substituentsindependently selected from the group consisting of hydrido, alkyl,cycloalkyl, heterocyclyl, aryl, and heteroaryl. The term “acyloxy”denotes an oxygen radical adjacent to an acyl group. The term“acylamino” denotes a nitrogen radical adjacent to an acyl group. Theterm “carboalkoxy” is defined as a carbonyl radical adjacent to analkoxy or aryloxy group. The term “carboxyamido” denotes a carbonylradical adjacent to an amino group. The term “carboxy” embraces acarbonyl radical adjacent to an alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclyl, aryl or heteroaryl group. The term “halo” is defined as abromo, chloro, fluoro or iodo radical. The term “thio” denotes a radicalcontaining a substituent group independently selected from hydrido,alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, attached to adivalent sulfur atom, such as, methylthio and phenylthio.

The term “alkyl” is defined as a linear or branched, saturated radicalhaving one to about ten carbon atoms unless otherwise specified.Preferred alkyl radicals are “lower alkyl” radicals having one to aboutfive carbon atoms. One or more hydrogen atoms of an alkyl can also bereplaced by a substituent group selected from acyl, amino, acylamino,acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy,nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl,heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl. Examples of alkylgroups include methyl, tert-butyl, isopropyl, and methoxymethyl.

The term “alkenyl” embraces linear or branched radicals having two toabout twenty carbon atoms, preferably three to about ten carbon atoms,and containing at least one carbon-carbon double bond. One or morehydrogen atoms of an alkenyl can also be replaced by a substituent groupselected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy,carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl,alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy,sulfoxy, and formyl. Examples of alkenyl groups include ethylenyl orphenyl ethylenyl.

The term “alkynyl” denotes linear or branched radicals having from twoto about ten carbon atoms, and containing at least one carbon-carbontriple bond. One or more hydrogen atoms of an alkynyl can also bereplaced by a substituent group selected from acyl, amino, acylamino,acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy,nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl,heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl. Examples of alkynylgroups include propynyl.

The term “aryl” denotes aromatic radicals in a single or fusedcarbocyclic ring system, having from five to twelve ring members. One ormore hydrogen atoms of an aryl may also be replaced by a substituentgroup selected from acyl, amino, acylamino, acyloxy, carboalkoxy,carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy,aryloxy, sulfoxy, and formyl. Examples of aryl groups include phenyl,naphthyl, biphenyl, and terphenyl. “Heteroaryl” embraces aromaticradicals which contain one to four hetero atoms selected from oxygen,nitrogen and sulfur in a single or fused heterocyclic ring system,having from five to fifteen ring members. One or more hydrogen atoms ofan heteroaryl may also be replaced by a substituent group selected fromacyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido,cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl.Examples of heteroaryl groups include pyridinyl, thiazolyl, thiadiazoyl,isoquinolinyl, pyrazolyl, oxazolyl, oxadiazoyl, triazolyl, and pyrrolylgroups.

The term “cycloalkyl” is defined as a saturated or partially unsaturatedcarbocyclic ring in a single or fused carbocyclic ring system havingfrom three to twelve ring members. One or more hydrogen atoms of acycloalkyl may also be replaced by a substituent group selected fromacyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido,cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl.Examples of a cycloalkyl group include cyclopropyl, cyclobutyl,cyclohexyl, and cycloheptyl.

The term “heterocyclyl” embraces a saturated or partially unsaturatedring containing zero to four hetero atoms selected from oxygen, nitrogenand sulfur in a single or fused heterocyclic ring system having fromthree to twelve ring members. One or more hydrogen atoms of aheterocyclyl may also be replaced by a substituent group selected fromacyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido,cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl.Examples of a heterocyclyl group include morpholinyl, piperidinyl, andpyrrolidinyl. The term “alkoxy” denotes oxy-containing radicalssubstituted with an alkyl, cycloalkyl or heterocyclyl group. Examplesinclude methoxy, tert-butoxy, benzyloxy and cyclohexyloxy. The term“aryloxy” denotes oxy-containing radicals substituted with an aryl orheteroaryl group. Examples include phenoxy. The term “sulfoxy” isdefined as a hexavalent sulfur radical bound to two or threesubstituents selected from the group consisting of oxo, alkyl,cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein at least one ofsaid substituents is oxo.

O-glycosylation-modulating compounds of the invention also include, butare not limited to any pharmaceutically acceptable salts, esters, orsalts of an ester of each compound. Examples of salts that may be used,which is not intended to be limiting include: chloride, acetate,hydrochloride, methansulfonate or other salt of a compound of Table 1 ora functional analog, derivative, variant, or fragment of the compound.

Derivatives of the compounds of Table 1 include compounds which, uponadministration to a subject in need of such administration, deliver(directly or indirectly) a pharmaceutically activeO-glycosylation-modulating (e.g. inhibiting) compound as describedherein. An example of pharmaceutically active derivatives of theinvention includes, but is not limited to, pro-drugs. A pro-drug is aderivative of a compound that contains an additional moiety that issusceptible to removal in vivo yielding the parent molecule as apharmacologically active agent. An example of a pro-drug is an esterthat is cleaved in vivo to yield a compound of interest. Pro-drugs of avariety of compounds, and materials and methods for derivatizing theparent compounds to create the pro-drugs, are known to those of ordinaryskill in the art and may be adapted to the present invention.

Analogs, variants, and derivatives of the compounds of the invention setforth in Table 1 may be identified using standard methods known to thoseof ordinary skill in the art. Useful methods involve identification ofcompounds having similar chemical structure, similar active groups,chemical family relatedness, and other standard characteristics. For thepurposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics 75^(th) Ed., inside cover, andspecific functional groups are defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in “OrganicChemistry”, Thomas Sorrell, University Science Books, Sausalito. 1999,the contents of which are incorporated herein by reference in theirentirety.

Using the structures of the compounds disclosed herein, one of ordinaryskill in the art is enabled to make predictions of structural andchemical motifs for analogs, variants, and/or derivatives that possesssimilar functions of the compounds disclosed in Table 1. Usingstructural motifs as search, evaluation, or design criteria, one ofordinary skill in the art is enabled to identify classes of compounds(functional derivatives, analogs, and/or variants of the OGT-modulatingcompounds) that possess the inhibitory function of the compoundsdisclosed herein. These compounds may be synthesized using standardsynthetic methods and tested for activity as described herein. Examplesof derivatives, analogs, and variants are known to those of skill in theart.

The invention also involves methods for determining the functionalactivity of O-glycosylation-modulating compounds described herein. Thefunction or status of a compound as an O-glycosylation-modulatingcompound can be determined according to assays known and those describedherein. For example, the enzyme assays described herein may be used toassess compounds for O-glycosylation-modulating ability. In addition,cell assays can be used to assess O-glycosylation-modulating ability ofcompounds. For example, cells can be contacted with a candidateO-glycosylation-modulating compound under conditions that produceO-glycosylation activity, and standard procedures can be used todetermine whether O-glycosylating enzyme activity is modulated (e.g.,inhibited or enhanced) by the compound and/or whether O-glycosylation ismodulated by the candidate compound. Such methods may also be utilizedto determine the status of analogs, variants, and derivatives asinhibitors of O-glycosylation enzyme activity and O-glycosylation.Although not intended to be limiting, examples of methods with which theability of an O-glycosylation-modulating compound to modulate or changeO-glycosylating enzyme activity and/or O-glycosylation can be tested,are purified enzyme assays, in vitro and in vivo assay systems providedherein in the Examples section.

Using such assays the level of O-glycosylating enzyme activity (e.g.binding and/or catalytic activity) and/or O-glycosylation can bemeasured in a system both before and after contacting the system with acandidate O-glycosylation-modulating compound as an indication of theeffect of the compound on the level of O-glycosylating enzyme activityand/or O-glycosylation. Secondary screens may further be used to verifythe efficacy of compounds identified as modulators of O-glycosylatingenzyme and/or O-glycosylation. Examples of initial displacement screensand secondary screens are provided in PCT application PCT/US2007/008806,which was filed Apr. 11, 2007. It will be understood by those ofordinary skill in the art that O-glycosylating enzymes andO-glycosylation substrates that may be used in methods and assays of theinvention include any suitable O-glycosylating enzymes and polypeptidesubstrates that can be O-glycosylated and that ones specificallydisclosed herein are exemplary O-glycosylating enzymes andO-glycosylation polypeptide substrates and are not intended to belimiting.

In addition, derivatives, analogs, and variants ofO-glycosylation-modulating compounds can be tested for theirO-glycosylating enzyme activity modulation and/or modulation ofO-glycosylation by using an activity assay (see examples). An example ofan assay method, although not intended to be limiting, is a kineticenzyme assay described herein. Such a kinetic assay may includecontacting a derivative, analog, or variant of anO-glycosylation-modulating compound with a glucosyltransferase enzymeand a sugar donor (e.g., a UDP-sugar) and a polypeptide that includes(i) an O-glycosylation site, (ii) a specific protease cleavage sitepositioned in the polypeptide such that the rate of cleavage of thepolypeptide by the specific protease at the specific protease cleavagesite is different when the polypeptide is O-glycosylated at theglycosylation site than when the polypeptide is not O-glycosylated atthe glycosylation site, and (iii) a detectable marker positioned in thepolypeptide such that detection of the detectable marker identifieswhether the polypeptide has been cleaved by the specific protease. Inaddition to the contacting step another step includes the addition tothe contacted polypeptide of the specific protease that cleaves at thespecific protease cleavage site of the polypeptide and the monitoring ofthe mixture of enzyme and the polypeptide for cleavage rate of thepolypeptide. In such an assay, the rate of cleavage is characteristic ofthe level of O-glycosylation of the glycosylation site.

Other cells and tissue-based assays may include contacting a tissue orcell sample with an O-glycosylation-modulating compound and determiningthe compound's modulatory activity as described herein. Contacting asimilar cell or tissue sample with an analog of theO-glycosylation-modulating compound, determining its activity, and thencomparing the two activity results can serve as a measure of theefficacy of the derivative, variant, and/or analog'sO-glycosylation-modulating activity.

In addition to the in vitro assays described above and the purifiedenzyme assays and other assays described in the Examples section, an invivo assay may be used to determine the functional activity ofO-glycosylation-modulating compounds described herein. In such assays,animal models of O-glycosylation-associated disease and/or disorders canbe treated with an O-glycosylation-modulating compound of the invention.O-glycosylation-modulation (e.g. inhibition) may be assayed usingmethods described herein, which may include labeling or imaging methods.Additionally, animals with and without O-glycosylation-modulatingcompound treatment can be examined for behavior and/or survival as anindication of the effectiveness and/or efficacy of the compound.Behavior may be assessed by examination of symptoms of abnormalO-glycosylating enzyme activity and/or abnormal O-glycosylation ofpolypeptides as described herein. These measurements can then becompared to corresponding measurements in control animals. For example,test and control animals may be examined following administration of anO-glycosylation-modulating compound of the invention. In someembodiments, test animals are administered an O-glycosylation-modulatingcompound of the invention and control animals are not. Any resultingchange in O-glycosylating enzyme activity and/or in the level ofO-glycosylation of polypeptides can then be determined for each type ofanimal using known methods in the art such as, but not limited to,methods described herein. Such assays may be used to compare levels ofO-glycosylating enzyme activity and/or O-glycosylation of polypeptidesin animals administered the candidate O-glycosylation-modulatingcompound to control levels of O-glycosylating enzyme activity and/orO-glycosylation of polypeptides in animals not administered theO-glycosylation-modulating compound as an indication that the putativeO-glycosylation-modulating compound is effective to alter (e.g. increaseor decrease) O-glycosylating enzyme activity and/or O-glycosylation ofpolypeptides. In other embodiments, a candidateO-glycosylation-modulating compound is administered to both a test andcontrol animal and the effect on O-glycosylating enzyme activity and/orO-glycosylation of polypeptides may be compared as a measure of theefficacy of the compound.

Once one or more O-glycosylation-modulating compounds are verified asinhibiting O-glycosylating enzyme activity and/or O-glycosylation ofpolypeptides using art-known assays or assays as described herein (e.g.,in Examples), further biochemical and molecular techniques may be usedto identify the targets of these compounds and to elucidate the specificroles that these target molecules play in the process of O-glycosylatingenzyme activity and/or O-glycosylation of polypeptides in associateddiseases and/or disorders. An example, though not intended to belimiting, is that the compound(s) may be labeled and contacted with acell to identify the host cell proteins with which these compoundsinteract. Such proteins may be purified, e.g., by labeling the compoundwith an immunoaffinity tag and applying the protein-bound compound to animmunoaffinity column.

Treatment

An O-glycosylation-modulating compound of the invention (e.g., anO-glycosylation inhibitor) may be used to treat a subject with anO-glycosylation-associated disease or disorder. As used herein, the term“treat” includes active treatment of a subject that has an OGT diseaseor disorder (e.g., a subject diagnosed with such a condition) and alsoincludes prophylactic treatment of a subject who is has not yet beendiagnosed. Compounds of the invention may be administeredprophylactically to a subject at risk of an O-glycosylation-associateddisorder or disorder. Determination of a subject at risk for anO-glycosylation-associated disease or disorder, and/or the determinationof a diagnosis of an O-glycosylation-associated disease or disorder in asubject, may be done by one of ordinary skill in the art using routinemethods.

An O-glycosylation-modulating compound of the invention may be deliveredto a cell using standard methods known to those of ordinary skill in theart. Various techniques may be employed for introducingO-glycosylation-modulating compounds of the invention to cells,depending on whether the compounds are introduced in vitro or in vivo ina host.

When administered, the O-glycosylation-modulating compounds (alsoreferred to herein as therapeutic compounds and/or pharmaceuticalcompounds) of the present invention are administered in pharmaceuticallyacceptable preparations. Such preparations may routinely containpharmaceutically acceptable concentrations of salt, buffering agents,preservatives, compatible carriers, and optionally other therapeuticagents.

The term “pharmaceutically acceptable” carrier means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredients. The characteristics ofthe carrier will depend on the route of administration.

The therapeutics of the invention can be administered by anyconventional route, including injection or by gradual infusion overtime. The administration may for example, be oral, intravenous,intraperitoneal, intrathecal, intramuscular, intranasal, intracavity,subcutaneous, intradermal, mucosal, transdermal, or transdermal.

The therapeutic compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. All methods include the step of bringing the compoundsinto association with a carrier which constitutes one or more accessoryingredients. In general, the compositions are prepared by uniformly andintimately bringing the therapeutic agent into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the therapeutic agent, whichis preferably isotonic with the blood of the recipient. This aqueouspreparation may be formulated according to known methods using thosesuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butane diol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono ordi-glycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables. Carrier formulations suitable for oral,subcutaneous, intravenous, intramuscular, etc. can be found inRemington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa.

Compositions suitable for oral administration may be presented asdiscrete units such as capsules, cachets, tablets, or lozenges, eachcontaining a predetermined amount of the therapeutic agent. Othercompositions include suspensions in aqueous liquors or non-aqueousliquids such as a syrup, an elixir, or an emulsion.

In some embodiments of the invention, an O-glycosylation-modulatingcompound of the invention may be delivered in the form of a deliverycomplex. The delivery complex may deliver the O-glycosylation-modulatingcompound into any cell type, or may be associated with a molecule fortargeting a specific cell type. Examples of delivery complexes includean O-glycosylation-modulating compound of the invention associated with:a sterol (e.g., cholesterol), a lipid (e.g., a cationic lipid, virosomeor liposome), or a target cell specific binding agent (e.g., anantibody, including but not limited to monoclonal antibodies, or aligand recognized by target cell specific receptor). Some complexes maybe sufficiently stable in vivo to prevent significant uncoupling priorto internalization by the target cell. However, the complex can becleavable under appropriate conditions within the cell so that theO-glycosylation-modulating compound is released in a functional form.

An example of a targeting method, although not intended to be limiting,is the use of liposomes to deliver an O-glycosylation-modulatingcompound of the invention into a cell. Liposomes may be targeted to aparticular tissue, such neuronal cells, or other cell type, by couplingthe liposome to a specific ligand such as a monoclonal antibody, sugar,glycolipid, or protein. Examples of neuronal cells include, but are notlimited to hippocampal cells. Such proteins include proteins orfragments thereof specific for a particular cell type, antibodies forproteins that undergo internalization in cycling, proteins that targetintracellular localization and enhance intracellular half life, and thelike.

For certain uses, it may be desirable to target the compound toparticular cells, for example specific neuronal cells, includingspecific tissue cell types, e.g. tissue-specific nervous system cells.In some embodiments, it may be desirable to target anO-glycosylation-modulating compound to another cell type, including, butnot limited to, cardiac cells, pancreatic cells, vascular cells, etc. Insuch instances, a vehicle (e.g. a liposome) used for delivering anO-glycosylation-modulating compound of the invention to a cell type(e.g. a neuronal cell, vascular cell, etc.) may have a targetingmolecule attached thereto that is an antibody specific for a surfacemembrane polypeptide of the cell type or may have attached thereto aligand for a receptor on the cell type. Such a targeting molecule can bebound to or incorporated within the O-glycosylation-modulating compounddelivery vehicle. Where liposomes are employed to deliver anO-glycosylation-modulating compound of the invention, proteins that bindto a surface membrane protein associated with endocytosis may beincorporated into the liposome formulation for targeting and/or tofacilitate uptake.

Liposomes are commercially available from Invitrogen, for example, asLIPOFECTIN™ and LIPOFECTACE™, which are formed of cationic lipids suchas N-[1-(2,3 dioleyloxy)-propyl]-N,N, N-trimethylammonium chloride(DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Methods formaking liposomes are well known in the art and have been described inmany publications.

The invention provides a composition of the above-described agents foruse as a medicament, methods for preparing the medicament and methodsfor the sustained release of the medicament in vivo. Delivery systemscan include time-release, delayed release or sustained release deliverysystems. Such systems can avoid repeated administrations of thetherapeutic agent of the invention, increasing convenience to thesubject and the physician. Many types of release delivery systems areavailable and known to those of ordinary skill in the art. They include,but are not limited to, polymer-based systems such as polylactic andpolyglycolic acid, poly(lactide-glycolide), copolyoxalates,polyanhydrides, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polycaprolactone. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Nonpolymer systems that are lipids including sterols such ascholesterol, cholesterol esters and fatty acids or neutral fats such asmono-, di- and tri-glycerides; phospholipids; hydrogel release systems;silastic systems; peptide based systems; wax coatings, compressedtablets using conventional binders and excipients, partially fusedimplants and the like. Specific examples include, but are not limitedto: (a) erosional systems in which the polysaccharide is contained in aform within a matrix, found in U.S. Pat. Nos. 4,452,775, 4,675,189, and5,736,152, and (b) diffusional systems in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-basedhardware delivery systems can be used, some of which are adapted forimplantation.

In one particular embodiment, the preferred vehicle is a biocompatiblemicroparticle or implant that is suitable for implantation into themammalian recipient. Exemplary bioerodible implants that are useful inaccordance with this method are described in PCT Internationalapplication no. WO 95/24929, entitled “Polymeric Gene Delivery System”.describes a biocompatible, preferably biodegradable polymeric matrix forcontaining an exogenous gene under the control of an appropriatepromoter. The polymeric matrix is used to achieve sustained release ofthe exogenous gene in the patient. In accordance with the instantinvention, the compound(s) of the invention is encapsulated or dispersedwithin the biocompatible, preferably biodegradable polymeric matrixdisclosed in WO 95/24929. The polymeric matrix preferably is in the formof a microparticle such as a microsphere (wherein the compound isdispersed throughout a solid polymeric matrix) or a microcapsule(wherein the compound is stored in the core of a polymeric shell). Otherforms of the polymeric matrix for containing the compounds of theinvention include films, coatings, gels, implants, and stents. The sizeand composition of the polymeric matrix device is selected to result infavorable release kinetics in the tissue into which the matrix device isimplanted. The size of the polymeric matrix device further is selectedaccording to the method of delivery which is to be used. The polymericmatrix composition can be selected to have both favorable degradationrates and also to be formed of a material which is bioadhesive, tofurther increase the effectiveness of transfer when the device isadministered to a vascular surface. The matrix composition also can beselected not to degrade, but rather, to release by diffusion over anextended period of time.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver agents and compounds of the invention of the invention to thesubject. Biodegradable matrices are preferred. Such polymers may benatural or synthetic polymers. Synthetic polymers are preferred. Thepolymer is selected based on the period of time over which release isdesired, generally in the order of a few hours to a year or longer.Typically, release over a period ranging from between a few hours andthree to twelve months is most desirable. The polymer optionally is inthe form of a hydrogel that can absorb up to about 90% of its weight inwater and further, optionally is cross-linked with multi-valent ions orother polymers.

In general, the agents and/or compounds of the invention are deliveredusing the bioerodible implant by way of diffusion, or by degradation ofthe polymeric matrix. Exemplary synthetic polymers which can be used toform the biodegradable delivery system include: polyamides,polycarbonates, polyalkylenes, polyalkylene glycols, polyalkyleneoxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinylethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone,polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof,alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, celluloseesters, nitro celluloses, polymers of acrylic and methacrylic esters,methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose,cellulose acetate, cellulose propionate, cellulose acetate butyrate,cellulose acetate phthalate, carboxylethyl cellulose, cellulosetriacetate, cellulose sulphate sodium salt, poly(methyl methacrylate),poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate),poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methylacrylate), poly(isopropyl acrylate), poly(isobutyl acrylate),poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethyleneglycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinylalcohols), polyvinyl acetate, poly vinyl chloride, polystyrene, andpolyvinylpyrrolidone.

Examples of non-biodegradable polymers include ethylene vinyl acetate,poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Examples of biodegradable polymers include synthetic polymers such aspolymers of lactic acid and glycolic acid, polyanhydrides,poly(ortho)esters, polyurethanes, poly(butic kid), poly(valeric acid),and poly(lactide-cocaprolactone), and natural polymers such as alginateand other polysaccharides including dextran and cellulose, collagen,chemical derivatives thereof (substitutions, additions of chemicalgroups, for example, alkyl, alkylene, hydroxylations, oxidations, andother modifications routinely made by those skilled in the art), albuminand other hydrophilic proteins, zein and other prolamines andhydrophobic proteins, copolymers and mixtures thereof. In general, thesematerials degrade either by enzymatic hydrolysis or exposure to water invivo, by surface or bulk erosion.

Bioadhesive polymers of particular interest include bioerodiblehydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell inMacromolecules, 1993, 26, 581-587, the teachings of which areincorporated herein by reference, polyhyaluronic acids, casein, gelatin,glutin, polyanhydrides, polyacrylic acid, alginate, chitosan,poly(methyl methacrylates), poly(ethyl methacrylates),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate).

Use of a long-term sustained release implant may be particularlysuitable for treatment of subjects with an established neurologicaldisorder or complication of diabetes as well as subjects at risk ofdeveloping a neurological disorder, insulin resistance, pre-diabetes,diabetes, or a complication of diabetes.

“Long-term” release, as used herein, means that the implant isconstructed and arranged to deliver therapeutic levels of the activeingredient for at least 7 days, and preferably 30-60 days, and mostpreferably months or years. The implant may be positioned at or near thesite of the neurological damage or the area of the brain or nervoussystem affected by or involved in the neurodegenerative disorder.Long-term release implants may also be used in non-neuronal tissues andorgans to allow regional administration of an OGT-modulating compound ofthe invention. Long-term sustained release implants are well known tothose of ordinary skill in the art and include some of the releasesystems described above.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

The preparations of the invention are administered in effective amounts.An effective amount is that amount of a pharmaceutical preparation thatalone, or together with further doses, stimulates the desired response.In the case of treating a disorder or condition that is associated withabnormal O-glycosylating enzyme activity and/or abnormal O-glycosylationof polypeptides, desired response is reducing the onset, stage orprogression of the abnormal O-glycosylating enzyme activity and/orO-glycosylation of polypeptides and associated effects. This may involveonly slowing the progression of the damage temporarily, although morepreferably, it involves halting the progression of the damagepermanently. An effective amount for treating abnormal O-glycosylatingenzyme activity and/or O-glycosylation of polypeptides is that amountthat alters (increases or reduces) the amount or level ofO-glycosylating enzyme activity and/or O-glycosylation of polypeptides,when the cell or subject is a cell or subject with anO-glycosylation-associated disease or disorder, with respect to thatamount that would occur in the absence of the active compound.

The invention involves, in part, the administration of an effectiveamount of an O-glycosylation-modulating compound of the invention. TheO-glycosylation-modulating compounds of the invention are administeredin effective amounts. Typically effective amounts of anO-glycosylation-modulating compound will be determined in clinicaltrials, establishing an effective dose for a test population versus acontrol population in a blind study. In some embodiments, an effectiveamount will be that amount that diminishes or eliminates anO-glycosylation-associated disease or disorder and its effects in acell, tissue, and/or subject. Thus, an effective amount may be theamount that when administered reduces the amount of cell and or tissuedamage and/or cell death from the amount that would occur in the subjector tissue without the administration of a O-glycosylation-modulatingcompound of the invention.

The pharmaceutical compound dosage may be adjusted by the individualphysician or veterinarian, particularly in the event of anycomplication. A therapeutically effective amount typically varies from0.01 mg/kg to about 1000 mg/kg, preferably from about 0.1 mg/kg to about200 mg/kg, and most preferably from about 0.2 mg/kg to about 20 mg/kg,in one or more dose administrations daily, for one or more days. It willbe recognized by those of skill in the art that some of theO-glycosylation-modulating compounds may have detrimental effects athigh amounts. Thus, an effective amount for use in the methods of theinvention may be optimized such that the amount administered results inminimal negative side effects and maximum O-glycosylation modulation.

The absolute amount will depend upon a variety of factors, including thematerial selected for administration, whether the administration is insingle or multiple doses, and individual subject parameters includingage, physical condition, size, weight, and the stage of the disease ordisorder. These factors are well known to those of ordinary skill in theart and can be addressed with no more than routine experimentation.

The pharmaceutical compounds of the invention may be administered alone,in combination with each other, and/or in combination with other drugtherapies that are administered to subjects withO-glycosylation-associated diseases. Additional drug therapies (foractive treatment and/or prophylaxis) that may be administered withpharmaceutical compounds of the invention include, art-known methods ofO-glycosylation-associated disorders such as treatments for diabetes,complications of diabetes, insulin resistance, neurodegenerativedisease, cancer, etc. Alternative drug therapies are known to those ofordinary skill in the art and are administered by modes known to thoseof skill in the art. The drug therapies are administered in amounts thatare effective to achieve the physiological goals (to reduce symptoms anddamage from O-glycosylation-associated disease or disorder in a subject,e.g. cell damage and/or cell death), in combination with thepharmaceutical compounds of the invention. Thus, it is contemplated thatthe alternative drug therapies may be administered in amounts that arenot capable of preventing or reducing the physiological consequences ofthe O-glycosylation-associated disease and/or disorder when the drugtherapies are administered alone, but which are capable of preventing orreducing the physiological consequences of an O-glycosylation-associateddisease and/or disorder when administered in combination with one ormore O-glycosylation-modulating compounds of the invention.

Examples of alternative drug therapies for regulating blood sugarlevels, e.g. therapies for pre-diabetes and/or diabetes, include oraltherapies with hypoglycemic agents an/or oral anti-diabetic agents,injectable therapies, and the like. Non-drug therapies for regulatingblood sugar level include, but are not limited to, diatetic and/orexercise control measures.

Diet and exercise alterations include, but are not limited to, reducingcaloric intake, and/or increasing fiber intake, and/or decreasing fatintake, and/or increasing exercise level.

Oral drug therapies for regulating blood sugar levels includehypoglycemic agents that may include, but are not limited to: Acarbose;Acetohexamide; Chlorpropamide; Darglitazone Sodium: Glimepiride;Glipizide; Glyburide, Repaglinide; Troglitazone; Tolazamide;Tolbutamide.

Oral drug therapies for regulating blood sugar levels includeantidiabetic agents that may include but are not limited to: Acarbose,Acetohexamide; Buformin; Butoxamine Hydrochloride; Camiglibose;Chlorpropamide; Ciglitazone; Englitazone Sodium; EtoforminHydrochloride; Gliamilide; Glibornuride; Glicetanile Gliclazide Sodium;Gliflumide; Glipizide; Glucagon; Glyburide; Glyhexamide; GlymidineSodium; Glyoctamide; Glyparamide; Insulin; Insulin, Dalanated; InsulinHuman; Insulin Human, Isophane; Insulin Human Zinc; Insulin Human Zinc,Extended; Insulin, Isophane; Insulin Lispro; Insulin, Neutral; InsulinZinc; Insulin Zinc, Extended; Insulin Zinc, Prompt; Linogliride;Linogliride Fumarate; Metformin; Methyl Palmoxirate; Palmoxirate Sodium;Pioglitazone Hydrochloride; Pirogliride Tartrate; Proinsulin Human;Repaglinide; Seglitide Acetate; Tolazamide; Tolbutamide; Tolpyrramide;Troglitazone; Zopolrestat.

Injectable therapies for regulating blood sugar levels may include, butare not limited to:

Fast-Acting Insulin:

Insulin Injection: regular insulin; Prompt Insulin Zinc Suspension;Semilente® insulin. These categories include preparations such as:Humalog® Injection; Humulin® R; Iletin II; Novolin R, Purified PorkRegular Insulin; Velosulin BR Human Insulin;

Intermediate-Acting Insulin:

Isophane Insulin Suspension: NPH insulin, isophane insulin; Insulin ZincSuspension Lente® Insulin. These categories include preparations suchas: Humulin® L; Humulin® R; Humulin® N NPH; Iletin® II, Lente®; Iletin®II, NPH; Novolin® L, Novolin® N, Purified Pork Lente® insulin, PurifiedPork NPH isophane isulin;

Intermediate and Rapid-Acting Insulin Combinations:

Human Insulin Isophane Suspension/Human Insulin Injection. This categoryincludes preparations such as: Humulin® 50/50; Humulin®70/30;Novolin®70/30

Long-Acting Insulin:

Protamine Zinc Insulin Suspension; Extended Insulin Zinc Suspension.These categories include preparations such as: Ultralente® Insulin,Humulin® U.

Diagnostic tests known to those of ordinary skill in the art may be usedto assess the level of O-glycosylating enzyme activity and/orO-glycosylation of polypeptides in a subject and the effects thereof,and to evaluate a therapeutically effective amount of a pharmaceuticalcompound administered. Examples of diagnostic tests are set forth below.A first determination of O-glycosylating enzyme activity and/or theeffects thereof in a cell and/or tissue may be obtained using one of themethods described herein (or other methods known in the art), and asecond, subsequent determination of the level of O-glycosylating enzymeactivity. A comparison of the O-glycosylating enzyme activity and/orO-glycosylation of polypeptides and/or the effects thereof on thesubject at the different time points may be used to assess theeffectiveness of administration of a pharmaceutical compound of theinvention as a prophylactic or an active treatment of theO-glycosylation-associated disease or disorder. Family history or prioroccurrence of an O-glycosylation-associated disease or disorder, even ifthe O-glycosylation-associated disease or disorder is absent in asubject at present, may be an indication for prophylactic interventionby administering a pharmaceutical compound described herein to reduce orprevent abnormal O-glycosylating enzyme activity and/or abnormalO-glycosylation of polypeptides.

An example of a method of diagnosis of abnormal O-glycosylating enzymeactivity and/or abnormal O-glycosylation of polypeptides that can beperformed using standard methods such as, but not limited to: imagingmethods, electrophysiological methods, blood tests, and histologicalmethods. Additional methods of diagnosis and assessment ofOGT-associated disease or disorders and the resulting cell death ordamage are known to those of skill in the art.

In addition to the diagnostic tests described above, clinical featuresof O-glycosylation-associated diseases and/or disorders can be monitoredfor assessment of O-glycosylating enzyme activity following onset of anO-glycosylation-associated disease or disorder. These features include,but are not limited to: assessment of the presence of cell damage, celldeath, neuronal cell lesions, brain lesions, organ lesions, abnormalcell growth, vascular damage, blood abnormalities, sugar processingabnormalities, and behavioral abnormalities. Such assessment can be donewith methods known to one of ordinary skill in the art, such asbehavioral testing, blood testing, and imaging studies, such asradiologic studies, CT scans, PET scans, etc.

Kits

The invention also provides a pharmaceutical kit comprising one or morecontainers comprising one or more O-glycosylation-modulating compoundsof the invention and/or formulations or compositions of the invention.The kit may also include instructions for the use of the one or moreO-glycosylation-modulating compounds or formulations of the inventionfor the treatment of an O-glycosylation-associated disease or disorder.The kits of the invention may also comprise one or more containerscontaining additional drugs for treating an O-glycosylation-associateddisease or disorder. The invention also includes in some aspects, kitsfor testing candidate compounds to assess their ability to inhibitO-glycosylating enzyme activity.

The invention will be more fully understood by reference to thefollowing examples. These examples, however, are merely intended toillustrate the embodiments of the invention and are not to be construedto limit the scope of the invention.

EXAMPLES Introduction

Preparation of polypeptide substrates and their use in assays forglycosylation modulators.

Example 1 Synthesis of Polypeptide Substrates

A polypeptide substrate was identified that satisfied threerequirements: 1) the peptide had to be a good substrate for O-LinkedN-acetylglucosaminyltransferase (OGT); 2) there must have been a largedifference in the rate of proteolysis between glycosylated andunglycosylated peptide; and 3) the composition/location of the FRET pairon the polypeptide must have yielded a large change in signal uponproteolysis.

To address the first requirement, a panel of polypeptides wassynthesized based on a truncated version of the CKII polypeptide [CKIIpeptide PGGSTPVSSNAMM (SEQ ID NO:20)]. The polypeptide was modified fromthe native sequence in several ways. First, a fluorescein was installedon the N-terminus during synthesis. Second, a lysine was added to theC-terminus so that a second dye or quencher could be installed byreaction with the appropriate N-hydroxysuccinimidyl ester. Thesemodifications allowed two different FRET approaches. The first approachrequired the installation of a coumarin derivative on the polypeptide.The second required installation of a quencher molecule, which measuredtotal fluorescence intensity. (See Examples 2 and 3). Finally, theresidues on either side of the glycosylated serine of the truncated CK2polypeptide were changed to phenylalanine, leucine or arginine tointroduce proteolytic cleavage sites for common proteases (such astrypsin, chymotrypsin and thermolysin) that were not present in thenative sequence. It was reasoned that cleavage adjacent to theglycosylation site would yield the largest difference in the rate ofproteolysis between glycosylated and native polypeptides. This series ofpolypeptides was synthesized and screened for OGT activity using byLC/MS. Peak integration (measured at 445 nm) was used to quantitativeproducts and reactants and mass spectrometry was used to confirm peakidentity. The results are presented in Table 2 and some of the peptides(peptide 4.1, 4.4, and 4.5 top to bottom) are illustrated in FIG. 2.

TABLE 2 Peptide derivatives of CK2. Mutated residuesare shown in italics and the glycolsylated serine is bold. Relative poly- rate of peptide mutation sequence glycosylation 4.1 wtFITC(Aha)STPVSSANMK 1 (SEQ ID NO: 7) 4.2 V → F FITC(Aha)STPF SSANMK   0.15 (SEQ ID NO: 8) 4.3 V → R FITC(Aha)STPR SSANMK 0 (SEQ ID NO: 9)4.4 S → F FITC(Aha)STPVS FANMK   0.4 (SEQ ID NO: 10) 4.5 S → RFITC(Aha)STPVS RANMK   0.7 (SEQ ID NO: 11)

The most striking result from this panel was that all polypeptideslacking the valine residue N-terminal to the glycosylated serine werepoor glycosyl acceptors. This result experimentally confirmed one aspectof OGT acceptor sequence specificity that had been recently proposedbased on data from the growing O-GlcNAcome—that a beta-branched aminoacid directly N-terminal to the glycosylated serine is favored in thepolypeptide acceptor sequence[25]. It also appeared that OGT toleratedsignificant variation in the position C-terminal to the glycosylatedresidue, accepting both phenylalanine and arginine with little loss ofactivity. Polypeptides 4.1, 4.4, and 4.5 were then derivatized at theε—NH₂ of the C-terminal lysine with the FRET donor7-diethylaminocoumarin-3-carboxylic acid (DEAC) using the commerciallyavailable NHS ester derivative, resulting in polypeptides 4.6-4.8. Thesemolecules were purified using RP-HPLC, enzymatically glycosylated, thenscreened for cleavage using a variety of proteases.

TABLE 3 DEAC modified polypeptide derivatives of CKII.Mutated residues are shown in italics and theglycolsylated serine is bold. peptide mutation sequence 4.6 WtFITC(Aha)STPVSSANMK(DEAC) (SEQ ID NO: 12) 4.7 S → F FITC(Aha)STPVSFANMK(DEAC) (SEQ ID NO: 13) 4.8 S → R FITC(Aha)STPVS RANMK(DEAC)(SEQ ID NO: 14)

Coumarin excitation (ex=400 nm) and subsequent emission (em=472 nm) inan intact polypeptide will efficiently excite a fluorescein analog(ex=485 nm), resulting in both coumarin (472 nm) and fluorescein (525nm) emission. When the polypeptide was cut, the amount of fluoresceinexcitation (and subsequent fluorescein emission) was greatly reduced,resulting in a large change in 535 nm/460 nm emission ratio. Oneadvantage of this readout was that the ratiometric response wasindependent of changes in volume and even small changes in FRET-probeconcentration[21].

The glycosylated and unglycosylated polypeptides were proteolysed withserial dilutions of trypsin, chymotrypsin, proteinase K, andthermolysin. All proteolysis led to a large (15-fold) and similar changein emission ratio, however only Proteinase K showed a large differencein the rate of proteolysis between glycosylated and unglycosylatedpolypeptides, cutting the unglycosylated polypeptides 4.6 and4.8˜100-500 times faster than the corresponding glycosylatedpolypeptides. The resulting assay window (defined as the area in betweenthe curve for the glycosylated and unglycosylated polypeptide, (FIG. 3)is very large and is roughly the same for both 4.6 and 4.8.

This was an unexpected and fortuitous result. It was anticipated thatcleavage would occur at the protease site designed into the polypeptide(i.e. after the arginine for trypsin or the phenylalanine forchymotrypsin) and therefore would not be directly adjacent to theglycosylated serine. This increase of one residue from the glycoserineresulted in a greatly reduced difference in proteolytic rate between theglycosylated and unglycosylated polypeptide, as can be seen in thetrypsin digest (FIG. 3). Although the substrate specificity forproteinase K is somewhat ambiguous, the enzyme is known to cleaveC-terminal to hydrophobic residues such as valine. The large differencein cleavage rate, indicative of cleavage directly adjacent to theglycosylated residue, as well as the fact that both 4.6 and 4.8 havesimilar assay windows led to the conclusion that proteinase K wascleaving after the valine, which was confirmed by analysis of thecleavage products by LC/MS.

From FIG. 3, the concentration of proteinase K that gives the largestassay window could be estimated; under the conditions above this valueis ˜50 ng/mL. Using this concentration of protease, several hundredpositive (BSA) and negative (OGT) control wells were analyzed and a Z′of 0.95 was calculated. This is the highest Z′ of any assay piloted atthe ICCB (Harvard University, Boston, Mass.)[26].

Before high-throughput screening, it was necessary to address severalconcerns regarding the use of the DEAC/HTC label pair. Autofluorescenceof library members can be problematic in screening as they lead to falsepositives. Unlike a fluorescence polarization assay, there is no way toidentify autofluorescent compounds during data analysis with a FRET orfluorescence intensity readout, so the effects of compoundautofluorescence are beneficially reduced during assay development. Theuse of DEAC was concerning for two reasons. First, the DEAC requiresexcitation at 400 nm, which is significantly blue-shifted compared tofluorescein excitation at 485 nm, indicating that autofluorescence couldbe more problematic than in the previous assay. The coumarin core, manyof which will likely be as fluorescent as the derivate used in the FRETpolypeptide, is present in 1.5% of the compounds in the ICCB libraries.Second, although the FRET polypeptide was used at quite high fluorophoreconcentrations (5 μM) during assay development, the planned final assayvolume (20 μL) and pin transfer volume of inhibitor (100 nL) correspondsto an average inhibitor concentration of ˜70 μM. Thus, compounds with aten-fold lower quantum yield and/or extinction coefficient than DEACwould likely yield significant interference.

To test for autofluorescence, 100 nL of ICCB plate 1364 (a mixture ofcommercially available known bioactive compounds) was pin transferred induplicate into 20 μL of a 5 μM solution of polypeptide 4.8 in buffer andthe plates were read at 460 nm and 535 nm. These compounds arestructurally diverse and represented the spectral characteristics of thelarger libraries. A scatter plot of the duplicate 535 nm/460 nm ratio(FIG. 4) revealed that roughly 5% of these compounds reproduciblyautofluoresced near 460 nm, and would be registered as positive in ahigh-throughput assay (since increased 460 nm emission will result in adecrease in the 535 nm/460 nm ratio).

Peptide Synthesis:

All polypeptides were purchased from Tufts University core facility(Boston, Mass.). The mass of crude product was verified and thepolypeptides were labeled under the following conditions: Polypeptidewas dissolved in 250 mM NaHCO₃ buffer, pH 8.2 at a 5 mM concentrationand the appropriate NHS-Ester (0.5 eq) was dissolved in DMSO (a volumeof 1/10 of the aqueous buffer was used). The DMSO solution was quicklyadded to the peptide and the reaction proceeded at room temperature for30 minutes. This was repeated 2 more times (four more times forpolypeptide 4.10). The reaction was monitored by LC/MS. LCMS conditionsfor polypeptides 4.1-4.8—Agilent Eclipse 5 μm XD8-C8 column, 0→100%CH₃CN+0.1% formic acid over 20 minutes. LCMS conditions for polypeptides4.9-4.10—Agilent Eclipse 5 μm XD8-C8 column, 0→100% CH₃CN+0.1% NH₄OHover 20 minutes. Each polypeptide was purified by preparative HPLC.Conditions for polypeptides 4.1-4.8 were: —Phenomonex Luna C18 5 μmcolumn, 0→100% CH₃CN+0.1% TFA over 40 minutes. Conditions forpolypeptides 4.9-4.10 were: —Phenomonex Luna C18 5 μm column, 0→100%CH₃CN+0.1% NH₄OH over 40 minutes. Products were confirmed by LC/MS.

Preparative Glycosylation of Labeled Polypeptides:

Purified FRET labeled polypeptides were added (20-50 μM finalconcentration) to assay buffer (50 mM Tris, 20 mM CaCl₂, 0.05% Tween-20,0.05% NP-40, pH=7.8), 200 μM UDP-GlcNAc, 0.05 units of alkalinephosphatase, and 5 μM OGT. Reactions were monitored using LCMS:conditions for polypeptides 4.1-4.8—Agilent Eclipse 5 μm XD8-C8 column,10→60% CH₃CN+0.1% formic acid over 20 minutes. LCMS conditions forpolypeptides 4.9-4.10—Agilent Eclipse 5 μm XD8-C8 column, 10→60%CH₃CN+0.1% NH₄OH over 20 minutes, and when complete were purified usingthe same column and conditions. Polypeptides 4.1-4.8 were quantitated byabsorption at 492 nm using an extinction coefficient of 86,000 cm⁻¹M⁻¹;polypeptide 4.9 was quantitated by absorption at 530 nm using anextinction coefficient of 26,000 cm⁻¹M⁻¹; polypeptide 4.10 wasquantitated by absorption at 530 nm using an extinction coefficient of52,000 cm⁻¹M⁻¹.

Fluorescence Resonance Energy Transfer (FRET)-Peptide Approach #1

This approach required the installation of a coumarin derivative on thepolypeptide. Given that the ICCB cherry pick limit of 0.2%,autofluorescence at a frequency of 1 compound in 1000 would result inroughly 50% of the compound picked would be false positives. Use of thecoumarin/FITC pair resulted in 50-fold more autofluorescence than thisupper limit, requiring adaptation of the assay. One possibility was toadd an additional read step in between compound transfer andproteolysis. Although this would successfully identify autofluorescentcompounds, it would also eliminate data for ˜5% of the library members,which include entire structural classes (FIG. 5). Due to concerns thatthe elimination of such a large number of compounds would adverselyaffect the number of OGT inhibitors identified, the FRET pair wasadjusted to reduce the number of compounds that autofluoresce.

Fluorescence Resonance Energy Transfer (FRET)-Peptide Approach #2

An alternative FRET strategy, the use of a donor/quencher pair wasemployed (FIG. 1). The readout using a quencher approach was rawfluorescence and was not ratiometric, thus the signal was subject towell-to-well volume variations introduced during liquid handling. Giventhe high Z′ and excellent signal-to-noise of this assay, it was reasonedthat a 3-5% variation from liquid handling would not be detrimental. Thesyntheses of the donor/quencher polypeptides 4.9 and 4.10 was undertakenand involved a one step coupling of QXL™ 520 acid-SE (Anaspec, San Jose,Calif.) with the ε-NH2 of the C-terminal lysine. Some solubility issueswere observed with polypeptides 4.6-4.8, prompting the synthesis of amore soluble variant, polypeptide 4.10. Improved polypeptide solubilityincreased the glycosylation rate by 2-3 fold. In addition to improvedsolubility, polypeptide 4.10 had two modified QXL™ 520 lysine residues.It was hypothesized that adding a second quencher would increase thechange in RFU when the polypeptide was cut. A small increase in windowsize was observed, but the difference was marginal. The assay windows ofboth polypeptides were similar to that of the corresponding DEAC/FITCpolypeptides 4.6 and 4.8, though the optimal proteinase K concentrationwas five-fold lower (˜10 ng/mL). Several hundred positive (BSA) andnegative (OGT) control wells were assayed with these polypeptides,yielding Z′ values of ˜0.90.

TABLE 4 DEAC modified polypeptide derivatives of CK2.Mutated residues are shown in italics and theglycolsylated serine is bold. peptide Mutation Sequence 4.9 S → RFITC(Aha)STPVS RANMK(QXL ™ 520) (SEQ ID NO: 15)  4.10 S → RFITC(Aha)STPVS RANMK(QXL ™ 520)R K(QXL ™ 520) (SEQ ID NO: 16)

Compound autofluorescence with polypeptides 4.9 and 4.10 was thenassessed using the same pilot experiment described above. The differencewas striking (FIG. 6). Using the FITC/QXL-520 labeled polypeptides, nocompound autofluorescence was observed with excitation at 485 nm. Notethat since the readout is total fluorescence, in this caseautofluorescence would lead to a reproducible increase in signal.

Another advantage of the quencher approach was assay linearity over theentire glycosylation range observed with both polypeptides 4.9 and 4.10(FIG. 7). This was not expected, and was not seen with in anypolypeptides 4.6 or 4.8. Linearity allows use of this assay for lowerthroughput applications that require greater sensitivity, such askinetics. It also makes a quantitative data workup possible, which wasexploited after the high-throughput screen (see below).

Conversion Target for Negative Controls

In kinetic assays it is important to establish an appropriate conversiontarget for the negative controls (in this case, the extent ofglycosylation in the absence of inhibitors) and determine theconcentration of reagents (i.e. OGT and both substrates) required toachieve this level of conversion. A reasonable conversion goal is 80%,since this will result in a large assay window but still ensure that thereaction is not run past completion, potentially missing weakinhibitors. The target time window to achieve this conversion was 2-4hours, which is sufficiently long to ensure that small variations in thetiming of liquid handling steps would not significantly affect theoutcome, but short enough to be practical. After significantoptimization, it was found that 4 μM of polypeptide 4.10, 20 μMUDP-GlcNAc, and 50-100 nM sOGT (depending on the enzyme batch) wouldreproducibly give 70-80% conversion in 2-4 hours. Because of the smallbut inherent variability, an aliquot of assay mix for each round of highthroughput screening was analyzed by LC/MS to determine the actualconversion for that run.

High Throughput Screening

With the final assay conditions established, 124,226 compounds werescreened in duplicate at the ICCB (Harvard University, Boston, Mass.)over two days; the highest throughput of any assay performed at the ICCBscreening facility[27]. Small molecule libraries from several commercialvendors were screened as well as known bioactive compounds, naturalproduct extracts, and molecules made using diversity oriented syntheticapproaches[28]. Included in this set were 52,026 of the 64,416 compoundsscreened using a donor displacement method such as that described in PCTapplication PCT/US2007/008806, which was filed Apr. 11, 2007. The vastmajority compounds screened were small molecules (average MW ˜350) thatwere optimized to have favorable physico-chemical properties such assolubility, decreased toxicity, and increased stability[29]. All of thecommercial compounds at the ICCB are plated at a stock concentration of5 mg/mL, which, given a 20 μL reaction volume, corresponds to a 25 μg/mLinhibitor concentration in each well. The format of most compound platesat the ICCB has the last two columns of each 384-well plate empty. Thisallowed each assay plate to have column of negative controls (runwithout inhibitor) and column of positive controls (run without sOGT),which allowed a Z′ to be calculated for each plate. These valuestypically ranged from 0.7-0.8.

Data workup began by calculating the average positive and negativecontrol values independently for both duplicates of each plate. Usingthe linear relationship between percent glycosylation and fluorescenceshown above (FIG. 7), the percent glycosylation was calculated for eachwell, with 0% corresponding to the average value of the positivecontrols and 100% corresponding to the average value of the negativecontrols. In actuality the range is roughly 0%480% glycosylatedpolypeptide since the reaction was not allowed to proceed to completion.However, this simplification was employed to facilitate the comparisonof different sets of plates. There were remarkably few compounds thatreproducibly appeared to inhibit OGT, which enabled hits to be selectedusing a very high cutoff. Compounds with <70% activity in one duplicateand <80% activity in the second duplicate were scored as hits. Evenusing this lax criterion, the total unfiltered hit set only numbered 84,corresponding to a hit rate of 0.065%.

Hit Validation for Both Assays

To validate the hits from the high throughput screen, a radiometricsecondary assay was used (adapted from that described in described inPCT Application PCT/US2007/008806, which was filed Apr. 11, 2007). All84 compounds were “cherry picked” and eight point IC₅₀ curves (withinhibitor concentration ranging from 125 μM to 970 nM) were generated.The CK23K polypeptide concentration was set at 250 μM, which is roughly1.1× the K_(m) value. The logic behind this was to mimic as much aspossible the sub-K_(n), FRET-peptide concentration present in theprimary assay, so that polypeptide competitive inhibitors would not beswamped out by the higher CK23K concentrations used to screen the donordisplacement assay. In addition, the buffer used in high throughputscreening, which had no NaCl and contains detergents, was used insecondary screening. Of the 84 compounds assayed, 38 (45%) showeddetectable inhibition at 125 μM, and 9 compounds (11%) had IC₅₀values≦20 μM. A relationship existed between hit potency in the primaryand secondary assay (FIG. 8), though there seemed to be a group ofstructurally unrelated weak validated inhibitors (IC₅₀ 125-250 μM) thatinhibited strongly in the primary assay.

The low primary hit rate and high hit validation percentage indicatedthat the assay had a remarkably low false positive rate, especiallyconsidering that the raw hits were not filtered. In particular, twotypes of false positives, promiscuous inhibitors and fluorescentcompounds, were almost entirely absent in this hit set. Given the largeoverlap of compounds between the two screens (52,026 compounds), it is acertainty that both types of compounds are present in the librariesscreened, but were engineered out of the hit set during assay design.Although not all of the validated hits had been analyzed for promiscuousinhibitory behavior, it was not likely that a sizable number of thesecompounds inhibited non-specifically. First, the structures of the hits(see below) had few features of promiscuous inhibitors[30, 31]. Shoichetand co-workers have recently proposed methods of greatly reducingpromiscuous hit in high throughput screening, the most important ofwhich is the use of detergent in the assay buffer[30, 32]. These dataobtained in these experiments corroborated that finding. Anotherimportant consideration is that any promiscuous inhibitor would likelyinhibit the protease (which is present at 1/100,000 the concentration ofOGT). Protease inhibition mimics polypeptide glycosylation and thus anegative well.

Fluorescent compounds were found in the hit set, but at a very lowfrequency (11 fluorescent compounds; 13% of total hits). As expected,none of these compounds showed inhibitory activity in the secondaryassay. Because both assays used a fluorescein-labeled probe molecule,the difference in the frequency of fluorescence interference was duesolely to the dye concentration, which is 50 nM in the donordisplacement high-throughput screen and 4 μM in the kinetic assay.Supporting this fact is that the structures of most of the 11 falsepositives have a similar, coumarin-2-thiazole/oxazole/imidazolestructure (FIG. 9), which is known to be highly fluorescent.

Of the 38 confirmed inhibitors, 23 (60%; 27% of the total hits) had IC₅₀values 5125 μM. Of these 23 compounds, four compounds contained reactiveor potentially reactive groups (FIG. 10). Five of the remaining 19 hitsclustered into two classes with shared structural elements. The first ofthese are compounds that contain a benzothiazole-6-carboxylic acidscaffold (FIG. 11). 94 compounds containing this core were assayed inthe primary screen. Insufficient data exists to comment on SAR trendsfor the Y position, but of the three inhibitors, the free carboxylicacid compound 4.11 (and smallest compound) is the most potent. It isnotable that no substitution was tolerated at the X position, even NH₂and CH₃ with Y═OH. The second clustering of compounds contains a corewith a β-amino acid. Although this grouping contains only two members,it is notable for several reasons. First, both compounds, 4.12 and 4.13(FIG. 12), have similar sterics at the α-amino position, and couldpotentially mimic the valine that was determined to be critical for OGTcatalysis previous work[25]. Second, there are only six compounds withthis conserved 3-amino-4-phenylbutanoic acid scaffold (shown at top ofFIG. 12) in the screened libraries, and only two of these six, 4.12 and4.13, have branched groups at the α-amino position. Third, 4.12 is themost potent OGT inhibitor discovered to date, with an IC₅₀=900 nM.Finally, 4.13 is the only inhibitor discovered in this assay that was ahit in the primary donor analog displacement assay (see PCT ApplicationPCT/US2007/008806, which was filed Apr. 11, 2007). This compound did notinhibit when screened in the secondary assay, perhaps because of thehigher polypeptide concentration used.

The remainder of the identified inhibitors appeared to have no conservedstructural features, however it should be noted that many of these hitscontained a free carboxylic acid (FIG. 13). Most of these compounds wereunique in the screened libraries, with few and often no strongly relatedcompounds.

Discussion

Although most of the compounds did not cluster, two structural scaffoldscould be identified. Almost all of the hits, singletons and clusters,were poorly represented in the ICCB commercial libraries and few analogswere commercially available. The syntheses of small chemical librariesare being explored to obtain SAR data around several of the compounds.

Another motivation guiding assay development was to add compounds thatinhibit through other binding modes to the set of available tools sinceUDP-GlcNAc competitive inhibition might affect normal O-GlcNAc processes(which occurs at low HSP flux and UDP-GlcNAc levels) before pathologicalones (occurs with increased flux)[33].

The assay described herein is the first kinetic assay that is readilyadaptable to high-throughput screening. A similar approach is used toscreen other glycosyltransferases that utilize polypeptide acceptorsubstrates, such as the enzymes involved in mucin biosynthesis and notchsignaling.

Assay Development and Validation Screening Protocol

384-well plates (Costar #3710) were filled using a liquid handling robotwith 10 μL of a mixture of 8 μM polypeptide 4.10, 40 μM UDP-GlcNAc, andbuffer (50 mM Tris, 20 mM CaCl₂, 0.05% Tween-20, 0.05% NP-40, pH=7.8,500 μM tris(hydroxypropyl)phosphine). Compound libraries were thentransferred to the assay plates using a 100 nL pin array and 10 μL of500 nM sOGT in buffer (50 mM Tris, 20 mM CaCl₂, 0.05% Tween-20, 0.05%NP-40, pH=7.8, 500 μM tris(hydroxypropyl)phosphine) was added using aliquid handling robot. The compounds were assayed at a concentration of25 μg/mL or ˜70 μM, assuming an average compound MW of 350. After 2-3hours, 20 μL of a 20 ng/mL proteinase K solution in buffer (50 mM Tris,20 mM CaCl₂, 0.05% Tween-20, 0.05% NP-40, pH=7.8) was added and allowedto react for 30-90 minutes. The plates were read using a Perkin-ElmerEnvision® microplate reader with a 480 nm excitation filter and 530 nmemission filter.

Hit Validation Protocols

Hits were examined using the secondary assay as described in PCTApplication PCT/US2007/008806, which was filed Apr. 11, 2007, with minormodifications. One microliter aliquots (5 mg/mL) of the hits wereobtained from the ICCB, and these were normalized to 5.0 mM with DMSO.These compounds were screened at 8 two-fold dilutions ranging from 125μM to 900 nM. The reaction mixture containing 250 μM polypeptide, 6.25μM UDP-¹⁴C-GlcNAc, ˜20-40 nM sOGT, and buffer (50 mM Tris, 20 mM CaCl₂,0.05% Tween-20, 0.05% NP-40, pH=7.8, 500 μMtris(hydroxypropyl)phosphine). Reactions were run for 20-30 minutes,then spotted on Whatman P81 phosphocellulose discs, washed three timesfor five minutes in 1% phosphoric acid, and counted by liquidscintillation counting. The IC₅₀ curve was fit to the following equationusing Prism 4 software (GraphPad Software, Inc., San Diego, Calif.).

Y=Ymin+(Ymax−Ymin)/(1+10̂((log(X)−log(IC50))*h))

where: X is the inhibitor concentration, Y is the reaction rate, and his the hill slope.

Example 2 An Additional Glycosylation Screening Assay

The sequelae associated with Clostridium difficile infection is causedmainly by the endocytosis of the bacterially produced toxins, ToxA(Genbank Accession No. P16154) and ToxB (Genbank Accession No. P18177).After these proteins are endocytosed into intestinal epithelium, thetoxin glycosylates many small GTPases at a known threonine residue.Polypeptides models on one of these GTPases, Rac 1 were synthesized. Rac1 has Genbank Accession No. P63000.

polypeptide 1 FITC-EYIPTVFDNK  (SEQ ID NO: 17) native sequencepolypeptide 2 FITC-EYRPTVFDNK I-->R (SEQ ID NO: 18) polypeptide 3FITC-EYIPTVDDNK F-->D (SEQ ID NO: 19)The glycosylated residues are bolded.

Each of these polypeptides is screened against thermolysin, ProteinaseK, and prolylendopeptidase (PEP). Thermolysin preferentially cleavessites with bulky and aromatic residues (Be, Leu, Val, Ala, Met, Phe) inposition P1′. Cleavage is favored with aromatic sites in position P1 buthindered with acidic residues in position P 1. Pro blocks when locatedin position P2′ but not when found in position P1. Thereforepolypeptides 1 and 3 are thermolysin candidates.

Proline-endopeptidase preferentially cleaves at Pro in position P1.Proline-endopeptidase may also accept Ala in position P1. With Pro inposition P1 the activity is blocked when another Pro is at position P1′.All polypeptides are candidates with cleavage with PEP, but polypeptide2 satisfies multiple criteria.

Proteinase K typically cleaves after hydrophobic amino acids, but itssubstrate specificity is somewhat ambiguous. All polypeptides arecandidates for Proteinase K. For expanded proteolysis rules, see: Keil,B. Specificity of proteolysis. Springer-Verlag Berlin-Heidelberg-NewYork, pp. 335. (1992).

Example 3 De-Glycosylation Assay

A glycosidase enzyme is contacted (under conditions suitable foractivity of the glycosidase enzyme) with a molecule comprising adetectably labeled polypeptide substrate having (i) a glycosylatedO-glycosylation site and (ii) a specific protease cleavage sitepositioned in the polypeptide such that the rate of cleavage of thepolypeptide by the specific protease at the specific protease cleavagesite is different when the polypeptide is O-glycosylated at theglycosylation site than when the polypeptide is not O-glycosylated atthe glycosylation site. The detectable label is positioned in thepolypeptide such that a change in the detectable label identifiescleavage of the polypeptide by the specific protease. The specificprotease that cleaves and the specific protease site in the polypeptideis added to the contacted polypeptide. The mixture is incubated underconditions suitable for activity of the specific protease. Cleavage ofthe polypeptide by the specific protease is monitored and the rate ofcleavage is characteristic of the level of de-glycosylation of theglycosylation site.

The assay is also run in a manner that includes contact of theglycosidase enzyme and the molecule that includes the detectably labeledpolypeptide substrate with a candidate compound. A reduction in cleavageby the protease in a sample that includes a candidate compound indicatesthat the compound inhibits the de-glycosylation (e.g. inhibits activityof the glyscosidase).

Example 4 Polypeptide N-acetylgalactosaminyltranferases (ppGalNAcTs)assays

There are multiple isoforms of the enzyme polypeptideN-acetylgalactosaminyltranferases (ppGalNAcTs). Activity of ppGalNAcTsare illustrated in FIG. 14. Using assay methods as described above,peptide substrates for ppGalNAcTs were prepared and used to assay toidentify modulators of ppGalNAcTs activity. Peptide substrates EPGPTEAPK(SEQ ID NO:25) and EDAVTPGPK (SEQ ID NO:26) were prepared (see FIG. 15)and utilized to assay for modulators of the enzymes. Each peptidesubstrate include a proteinase K cleavage site. The identified compoundSEQ ID NO:25 is a general substrate for ppGalNAcTs and identifiedcompound SEQ ID NO:26 is a selective substrate for the enzymeppGalNAcT1. Assays were run and alterations in the rate and/or amount ofcleavage by proteinase K was determined. The cleavage productsillustrated in FIG. 16 show that GTF+ inhibitor resulted in cleavage byproteinase K. Little or no cleavage was observed without the presence ofa ppGalNacT inhibitor. A graphic representation of the assay of cleavageof the peptide substrate under conditions with or without glycosylationof the substrate is presented in FIGS. 17A and B which show thatcleavage of unglycosylated substrate greatly exceeds that ofglycosylated substrate. The difference in cleavage permitted use of theassay to identify compounds that inhibit glycosylation of the substrateby ppGalNAcTs. Additional inhibitory compounds identified using theassay include Compound 24 and Compound 27 (see Table 1).

Using the assay, compounds that inhibit ppGalNAcTs were identified.Identified compounds included compounds 24-27 (see Table 1).

It was determined that the assay could also be used to identify isoformselective inhibitors. For example, FIG. 18 A shows results from ageneral ppGalNAcT inhibitor (compound 24), which inhibited bothppGalNAcT1 and ppGalNAcT2. FIG. 18 B shows results from compound 25,which was a more selective inhibitor for ppGalNAcT2 than for ppGalNAcT1.Additional small molecule inhibitors of ppGalNAcT are shown in FIG. 19.FIG. 20 shows the effect of an identified small molecule inhibitor oncrystal growth of ppGalNAcT2. The presence of the inhibitor disruptedcrystal growth of the ppGalNAcT2.

The assays results demonstrate an additional kinetic assay that can beused to identify inhibitors of O-linked glycosylation. The resultsindicated that the O-linked glycosylation assays of the invention areuseful for identifying modulators of O-linked glycosylation that iscarried out by multiple different O-linked glycosylating enzymes andthat the assays could be used to identify isoform-specificglycosyltranferase (Gts) inhibitors. Thus, the screens could be used toassay Gtfs from different Gts classes.

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EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references, including patent documents, disclosed herein areincorporated by reference in their entirety.

What is claimed is:
 1. An isolated molecule comprising a cleavabledetectably labeled polypeptide substrate having a) an O-glycosylationsite and b) a specific protease cleavage site positioned in thepolypeptide such that the rate of cleavage by a specific protease at thespecific protease cleavage site is different when the polypeptide isglycosylated at the O-glycosylation site than when the polypeptide isnot glycosylated at the O-glycosylation site, wherein the detectablelabel is positioned in the polypeptide such that a change in thedetectable label identifies cleavage of the polypeptide by the specificprotease at the specific protease cleavage site.
 2. The isolatedmolecule of claim 1, wherein the polypeptide O-glycosylation site is aserine or a threonine residue.
 3. The isolated molecule of claim 2,wherein the serine or threonine is positioned within 1, 2, 3, or 4 aminoacids of the specific protease cleavage site.
 4. The isolated moleculeof claim 1, wherein the specific protease cleavage site is a single siteand there is only one specific protease cleavage site.
 5. The isolatedmolecule of claim 1, wherein the specific protease cleavage site ispositioned on the N-terminal side of the O-glycosylation site.
 6. Theisolated molecule of claim 1, wherein the specific protease cleavagesite is positioned on the C-terminal side of the O-glycosylation site.7. The isolated molecule of claim 1, wherein the detectable labelcomprises a fluorescent, enzyme, radioactive, metallic, biotin,chemiluminescent, or bioluminescent molecule.
 8. The isolated moleculeof claim 1, wherein the detectable label is a fluorescent moiety.
 9. Theisolated molecule of claim 8, wherein the change in the detectable labelcomprises a change in fluorescence of the fluorescent moiety.
 10. Theisolated molecule of claim 1, wherein the detectable label is a FRETdonor and a FRET acceptor pair.
 11. The isolated molecule of claim 10,wherein the FRET donor and acceptor pair is7-diethylaminocoumarin-3-carboxylic acid (DEAC) and fluoresceinisothiocyanate; [2-(1-sulfonyl-5-naphthyl)-aminoethylamide] EDANS and4,4-dimethylazobenzene-4′carbonyl (DABCYL); fluorescein isothiocyanateand tetramethylrhodamine (TMR); or Tryptophan or tyrosine anddinitrophenyl moieties.
 12. The isolated molecule of claim 8, whereinthe fluorescent moiety is a Cy5.5 emitter or FITC, Texas red,tetramethylrhodamine (TMR), AlexaFluor dyes, HiLyte Fluorophores, or[2-(1-sulfonyl-5-naphthyl)-aminoethylamide] EDANS.
 13. The isolatedmolecule of claim 1, wherein the polypeptide further comprises aquenching moiety.
 14. The isolated molecule of claim 13, wherein thequenching moiety is QXL-520™, BHQ3, Iowa black,4,4-dimethylazobenzene-4′carbonyl (DABCYL), BHQ1, BHQ10, QXL-570,QXL-620, dinitrophenyl (DNP) containing groups, or QSY-7, 9-21, or 35.15. The isolated molecule of claim 1, wherein the polypeptide comprisesan amino acid sequence selected from the group consisting of: STPVSSANMK(SEQ ID NO:1), STPVSFANMK (SEQ ID NO:2), STPVSRANMK (SEQ ID NO:3),EYIPTVFDNK (SEQ ID NO:4), EYRPTVFDNK (SEQ ID NO:5), EYIPTVDDNK (SEQ IDNO:6), EPGPTEAPK (SEQ ID NO:25), and EDAVTPGPK (SEQ ID NO:26). 16-22.(canceled)
 23. The isolated molecule of claim 1, wherein the specificprotease cleavage site is a proteinase K cleavage site, a trypsincleavage site, a chymotrypsin cleavage site, a thermolysin cleavagesite, Staphylococcal peptidase I cleavage site, Proline-endopeptidasecleavage site, Pepsin cleavage site, Glutamyl endopeptidase cleavagesite, Factor Xa cleavage site, Granzyme B Lysyl endopeptidase cleavagesite, Asp-N Endopeptidase cleavage site, or enterokinase cleavage site.24-36. (canceled)
 36. A kit for identifying an agent that modulatesO-glycosylation, the kit comprising a package housing a first containercontaining a polypeptide of claim 1, and instructions for using thepolypeptide to identify modulators of O-glycosylation.
 37. The kit ofclaim 36, further comprising a second container containing the specificprotease that cleaves at the specific protease site of the polypeptide.38. The kit of claim 36, wherein the polypeptide comprises the aminoacid sequence set forth as one of SEQ ID NOs:1-6, 25, or
 26. 39-69.(canceled)